WO2012154368A1 - Precipatable peptipes - Google Patents

Precipatable peptipes Download PDF

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Publication number
WO2012154368A1
WO2012154368A1 PCT/US2012/033293 US2012033293W WO2012154368A1 WO 2012154368 A1 WO2012154368 A1 WO 2012154368A1 US 2012033293 W US2012033293 W US 2012033293W WO 2012154368 A1 WO2012154368 A1 WO 2012154368A1
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WIPO (PCT)
Prior art keywords
seq
pbrc
amino acid
pbrts
variant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/US2012/033293
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French (fr)
Inventor
Scott Banta
Oren SHUR
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Columbia University in the City of New York
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Columbia University in the City of New York
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Application filed by Columbia University in the City of New York filed Critical Columbia University in the City of New York
Publication of WO2012154368A1 publication Critical patent/WO2012154368A1/en
Priority to US14/052,367 priority Critical patent/US9550805B2/en
Anticipated expiration legal-status Critical
Priority to US15/377,243 priority patent/US10358461B2/en
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C07K1/303—Extraction; Separation; Purification by precipitation by salting out
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/24—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a MBP (maltose binding protein)-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • C07K2319/92—Fusion polypeptide containing a motif for post-translational modification containing an intein ("protein splicing")domain

Definitions

  • Rapid protein purification is an important requirement in many bioengineering applications where significant amounts of time are currently spent purifying proteins from heterogeneous samples.
  • bioseparation There are currently a number of approaches for performing bioseparation, but these approaches are expensive, time consuming, can require specialized treatments.
  • a variety of approaches currently exist for purifying recombinant proteins such as using a poly-histidine tag, glutathione S-transferase (GST) fusions or fusion to an elastin-like peptide (ELP).
  • GST glutathione S-transferase
  • ELP elastin-like peptide
  • a fusion protein can be precipitated from solution by increasing the temperature of the sample (Banki, et al., Nat Meth, vol. 2, no. 9, pp. 659-662, 2005; Fong et al, Trends in Biotechnology, vol. 28, no. 5, pp. 272-279, May. 2010).
  • GST glutathione S-transferase
  • ELP elastin-like peptide
  • the invention relates to a precipatable beta roll cassette (PBRC) comprising one or more beta roll tags (PBRTs) wherein the one or more PBRTs comprise the amino acid sequence of SEQ ID NO: 1
  • the invention relates to a precipatable beta roll cassette (PBRC) comprising one or more beta roll tags (PBRTs) wherein the one or more PBRTs are independently any of: (a) a polypeptide having the amino acid sequence of SEQ ID NO: 1, or (b) a polypeptide having the amino acid sequence of any of SEQ ID NOs: 25-1337
  • PBRC precipatable beta roll cassette
  • PBRTs beta roll tags
  • the invention relates to a PBRC linked purification moiety comprising a precipatable beta roll cassette (PBRC) comprising one or more beta roll tags (PBRTs) wherein the one or more PBRTs are independently any of: (a) a polypeptide having the amino acid sequence of SEQ ID NO: 1, or (b) a polypeptide having the amino acid sequence of any of SEQ ID NOs 25-1337, (c) a polypeptide comprising the amino acid sequence GXXXXXXX, wherein, (i) the X at position 2 is an amino acid selected from the group consisting of glycine, asparagine or aspartic acid, and (ii) the X at position 3 is an amino acid selected from the group consisting of alanine, serine, glycine, aspartic acid, glutamic acid, leucine or asparagine, and (iii) the X at position 4 is an amino acid selected from the group consisting of glycine
  • the invention relates to a PBRC linked purification moiety comprising a precipatable beta roll cassette (PBRC) comprising one or more beta roll tags (PBRTs) wherein the one or more PBRTs are independently any of: (a) a polypeptide having the amino acid sequence of SEQ ID NO: 1, or (b) a polypeptide having the amino acid sequence of any of SEQ ID NOs 25-1337, (c) a polypeptide comprising the amino acid sequence GXXXXXXX, wherein, (i) the X at position 2 is an amino acid selected from the group consisting of glycine, asparagine or aspartic acid, and (ii) the X at position 3 is an amino acid selected from the group consisting of alanine, serine, glycine, aspartic acid, glutamic acid, leucine or asparagine, and (iii) the X at position 4 is an amino acid selected from the group consisting of glycine
  • the cleavage site is selected from the group comprising an intein cleavage site, a Factor Xa cleavage site, a thrombin cleavage site, an enterokinase cleavage site, or a signal peptidase cleavage site.
  • the invention relates to a polypeptide comprising a PBRC linked purification moiety comprising a precipatable beta roll cassette (PBRC) comprising one or more beta roll tags (PBRTs) wherein the one or more PBRTs are independently any of: (a) a polypeptide having the amino acid sequence of SEQ ID NO: 1, or (b) a polypeptide having the amino acid sequence of any of SEQ ID NOs 25-1337, (c) a polypeptide comprising the amino acid sequence GXXXXXXX, wherein, (i) the X at position 2 is an amino acid selected from the group consisting of glycine, asparagine or aspartic acid, and (ii) the X at position 3 is an amino acid selected from the group consisting of alanine, serine, glycine, aspartic acid, glutamic acid, leucine or asparagine, and (iii) the X at position 4 is an amino acid selected from the group
  • the invention relates to a nucleic acid encoding any of the polypeptides described herein.
  • the invention relates to a method for purifying a PBRC linked purification moiety, the method comprising (a) expressing the PBRC linked purification moiety in an expression system, (b) collecting the PBRC linked purification moiety in a first medium, (c) adding Ca2+ to the first medium so as to induce precipitation of PBRC linked purification moiety, (d) removing unprecipiated material from the medium from the precipitated PBRC linked purification moiety, (e) resuspending the PBRC linked purification moiety in a second medium having a lower than the free Ca2+ concentration than the free Ca2+ concentration obtained after step (c).
  • a calcium chelator is added to the second medium of step (e). In certain embodiments, steps (c) to (e) are repeated one or more times. In certain embodiments, the method further comprises a step of removing precipitated material between step (b) and step (c). In certain embodiments, the PBRC comprises a cleavage site between the PBRC and the purification moiety. In certain embodiments, the method further comprises steps of: (i) cleaving the PBRC linked purification moiety so as to separate the purification moiety from the PBRC, (ii) adding Ca2+ to the medium so as to induce precipitation of the PBRC, and (iii) isolating the unprecipiated purification moiety. In certain embodiments, the cleavage site is an intein cleavage site.
  • the invention relates to an expression vector comprising, as arranged from 5' to 3', a promoter, a nucleic acid sequence encoding the PBRC of any of claims 1-4, and at least one cloning site.
  • the invention relates to an expression vector comprising, as arranged from 5' to 3', a promoter, at least one cloning site, and a nucleic acid sequence encoding the PBRC of any of claims 1-4.
  • the invention relates to an expression vector comprising, as arranged from 5' to 3', a promoter, at least one cloning site, a nucleic acid sequence encoding the PBRC of any of claims 1-4 and at least a second cloning site.
  • Figure 1 shows a SDS-PAGE gel showing purification of a molecule comprising a precipatable beta-roll tag.
  • Figure 1 A shows purification of precipitatable maltose binding protein comprising a precipitatable beta roll tag and an enterokinase cleavage site (MBP- PBRT). Total lysate is shown in lane 1 and lanes 2-7 are precipitation/ wash cycles. After two cycles, the sample consists nearly only of the MBP fusion protein with the precipitating tag attached (MBP-PBRT). Recovery is nearly 100% of the expressed protein.
  • Figure IB shows SDS-PAGE analysis of purified MBP-PBRT and MBP-PBRT subjected to digestion of the enterokinase digestion site ( Figure IB). Lane 1 shows purified MBP-PBRT, lane 2 shows supernatant after overnight digest and lane 3 shows the pellet.
  • Figure 2 shows a SDS-PAGE gel showing a successful purification of a polypeptide comprising a 5 or 17 repeat C-capped precipatable beta-roll tags. Lanes (from left to right): 1. MBP-5cap lysate, 2. MBP-5cap supernatant, 3. MBP-5C resuspended precipitate. The 4-6 lanes are the same expect with the capped 17 repeat construct.
  • Figure 3 is circular dichroism data (CD) showing precipitation of a polypeptide comprising a 17 repeat C-capped precipatable beta-roll tag
  • Figure 4 shows a GGXGXDXXX (SEQ ID NO: 2) sequence heat map. The heat map was determined by using BLAST to find beta roll sequences similar to the metalloprotease of S. marcescens and then quantifying the frequency of amino acids at each of the nine positions after beta roll sequences were identified. See SEQ ID NO: 1343.
  • Figure 5 shows a schematic illustration of the corkscrew configuration of tandem Ca2+ binding sequences. The figure shows a crystal structure image of the beta roll domain from the metalloprotease of S. marcescens (1SAT in the Protein Databank). Alternating 9- amino acid repeats are highlighted in green and red. Coordinated calcium ions are in white. The image represents 5 repeats of beta roll sequence. While there is no crystal structure for the adenylate cyclase beta roll domain, the high degree of sequence similarity to the consensus beta roll indicates that the adenylate cyclase beta roll domain is similar.
  • Figure 6 shows a schematic illustration of the corkscrew configuration of tandem Ca2+ binding sequences from a different angle than shown in Figure 5.
  • the 6 th residue binds the calcium ion.
  • the 7th and 9th residues of each repeat are those that face outwards.
  • the 8 th residue is buried in the hydrophobic core. These residues are threonine and tyrosine, respectively in SEQ ID NO: 1.
  • Figure 7 shows the full crystal structure of the metalloprotease of S. marcescens.
  • the black spheres indicate the position of the calcium ions within the beta roll domain.
  • Figure 8 shows a characterization of beta roll distribution, sequence deviation and number of repeats.
  • Figure 8 A shows a distribution of beat roll lengths as frequency plotted against the number of beta roll repeats.
  • Figure 8B shows beta roll sequence deviation from consensus by position plotted as proportion deviation as a function of distance from terminus.
  • Figure 8C shows a probability of deviation from consensus plotted as probability as a function of the number of beta roll repeats.
  • Figure 8D shows amount of beta roll in overall protein plotted as the number of beta roll residues as a function of the number of total residues.
  • Figure 9 shows an exemplary protocol for purification of a polypeptide comprising a precipatable beta roll tag.
  • the images depict precipitation of maltose binding protein fused to seventeen repeats of a PBRT.
  • the PBRC comprises 17 repeats of the amino acid sequence of SEQ ID NO: 1.
  • the PBRC does not comprise a capping sequence.
  • Figure 10 shows purification of a maltose binding protein/PBRT/green fluorescent protein fusion (MBP-PBRT-GFP).
  • Figure 10A shows a precipitation and resuspension of the MBP-PBRT-GFP polypeptide.
  • Figure 10B shows SDS-PAGE of multiple precipitation wash cycles.
  • the lanes of the SDS gel are as follows: lane 1 - ladder; lane 2 - clarified lysate; lane 3 - precipitation in 25 mM calcium followed by three washes; lane 4 - precipitation in 50 mM calcium followed by three washes; lane 5 - precipitation in 75 mM calcium followed by three washes; lane - 6 precipitation in 100 mM calcium followed by three washes. All lanes are normalized in terms of concentration. Recovery percentage can be estimated by comparing the band intensity in lane 2 to subsequent lanes.
  • Figure 11 shows non-limiting examples of polypeptides comprising a PBRT suitable for use with the methods described herein.
  • a target polypeptide can be produced as a fusion protein in frame with a PBRC.
  • the fusion protein comprising the target protein and the PBRC can further comprise a specific cleavage site (e.g. an intein cleavage site or an enterokinase cleavage site) between the target protein sequence and the PBRC sequence.
  • cleavage at the cleavage site can be used to separate the PBRC from the target polypeptide.
  • the beta-roll domain is a right-handed beta helix found in a number of proteins.
  • the consensus sequence for beta-roll peptides is tandem repeats of the 9 amino acid sequence GGXGXDX(L/F/I)X (SEQ ID NO: 24).
  • the conformation aligns to adopt the helical turns.
  • Two repeats of the sequence are required to make a complete helical turn and each of these turns binds a calcium atom.
  • the peptide exists in a disordered conformation. Therefore the ⁇ -roll domain exhibits natural allosteric regulation.
  • a synthetic version of the ⁇ -roll peptide has been produced with 8 repeats of GGSGNDNLS (SEQ ID NO: 1338) and this peptide was found to bind calcium and fold into the ⁇ -roll structure (Lilie et al, FEBS Lett 470 (2), 173 (2000)). The domain is capable of reversibly unfolding upon removal of the calcium.
  • Beta roll sequences are known to play a role in secretion as part of the bacterial Type I secretion system (Davidson, et al., Microbiol. Mol. Biol. Rev. 72 (2008), pp. 317-364; Holland et al, Mol. Membr. Biol. 22 (2005), pp. 29-39; Chenal, et al, J. Biol. Chem.
  • the precipatable-beta roll tags and precipatable-beta roll cassettes described herein are class of designed peptides which possess the ability to reversibly precipitate in response to calcium ions.
  • the invention described herein relates to the surprising finding that PBRCs (e.g. PBRTs repeats of sequence GGAGNDTLY (SEQ ID NO: 1)) undergo reversible precipitation upon calcium binding.
  • PBRCs e.g. PBRTs repeats of sequence GGAGNDTLY (SEQ ID NO: 1)
  • attachment of a PBRC to a second molecule e.g. attachment to a protein as a fusion protein comprising an in- frame
  • the invention described herein relates to the use of calcium concentration changes at room temperature to induce precipitation of recombinant molecules comprising a precipatable beta-roll tag.
  • the PBRCs described herein are also suitable for purifying non-peptide purification moieties of widely varying types, including, for example, lipids, oligonucleotides and carbohydrates, small organic or inorganic molecules, proteins, single-stranded or double-stranded oligonucleotides, polynucleotides.
  • applications for the methods and compositions described herein include, but are not limited to, the purification of recombinant proteins the removal of target proteins from a sample, and detection of compounds for diagnostic purposes.
  • the invention also extends to the antibodies that specifically bind to a PBRT or a PBRC and the methods for using the PBRTs and PBRCs described herein.
  • the PBRTs and PBRCs described herein can undergo a reversible Ca2+ binding dependent transition wherein they are structurally disordered and highly soluble in a medium below a Ca2+ concentration (or free Ca2+) transition concentration, but exhibit a disorder to order phase transition when the Ca2+ or free Ca2+ concentration is raised above the Ca2+ (or free Ca2+) transition concentration.
  • the disorder to order phase transition leads to precipitation of the PBRTs or PBRCs. Precipitation of PBRC can be used to remove and isolated them from solution (e.g. by centrifugation).
  • the invention described herein relates to a PBRC which functions reversible Ca2+ precipatable tag when linked to a purification moiety of interest.
  • the methods described herein can be used to induce precipitation of the PBRC linked purification moiety. Because the transition concentration dependent phase transition is reversible, the PBRT and PBRC can be resolubilized in a medium having a Ca2+ concentration (or free Ca2+) below the transition concentration. In certain embodiments, this can be accomplished by introducing medium having reduced, or no Ca2+, or by removing, or chelating Ca2+ from the medium. When the precipitate is resuspended in calcium-free buffer or in a buffer comprising a calcium ion chelator (e.g. EGTA or EDTA), the precipitate resuspends into solution.
  • a calcium ion chelator e.g. EGTA or EDTA
  • PBRT precipatable-beta roll tag
  • GGAGNDTLY SEQ ID NO: 1
  • a PBRT refers to an amino acid sequence having the amino acid sequence GXXXXXXX (SEQ ID NO: 1343), wherein (a) the X at position 2 is an amino acid selected from the group consisting of glycine, asparagine or aspartic acid, and (b) the X at position 3 is an amino acid selected from the group consisting of alanine, serine, glycine, aspartic acid, glutamic acid, leucine or asparagine, and (c) the X at position 4 is an amino acid selected from the group consisting of glycine or alanine, and (d) the X at position 5 is an amino acid selected from the group consisting of asparagine, aspartic acid, alanine, or serine, and (e)
  • GDNASDLFS SEQ ID NO: 25 GGGGNDTLI SEQ ID NO: 463 GGAGDDTLV SEQ ID NO: 901
  • GGGGNDTLY SEQ ID NO: 31 GGAGDDTLI SEQ ID NO: 469 GGGGDDTLV SEQ ID NO: 907
  • GDAGDDTLY SEQ ID NO: 39 GDGGDDTLI SEQ ID NO: 477 GDAGADTLV SEQ ID NO: 915
  • GNGGDDTLY SEQ ID NO: 44 GNAGADTLI SEQ ID NO: 482
  • GGAGADTLY SEQ ID NO: 49 GGGGADTLI SEQ ID NO: 487
  • GDAGADTLY SEQ ID NO: 51 GDGGADTLI SEQ ID NO: 489 GDAGNNTLV SEQ ID NO: 927
  • GDSGADTLY SEQ ID NO: 54 GDDGADTLI SEQ ID NO: 492 GDSGNNTLV SEQ ID NO: 930
  • GNDGADTLY SEQ ID NO: 59 GNSGNNTLI SEQ ID NO: 497 GNDGNNTLV SEQ ID NO: 935
  • GGAGNNTLY SEQ ID NO: 61
  • GGGGNNTLI SEQ ID NO: 499
  • GGAGDNTLV SEQ ID NO: 937
  • GNAGNNTLY SEQ ID NO: 62 GNGGNNTLI SEQ ID NO: 500 GNAGDNTLV SEQ ID NO: 938
  • GDAGNNTLY SEQ ID NO: 63
  • GDGGNNTLI SEQ ID NO: 501
  • GDAGDNTLV SEQ ID NO: 939
  • GGSGNNTLY SEQ ID NO: 64 GGDGNNTLI SEQ ID NO: 502 GGSGDNTLV SEQ ID NO: 940
  • GDSGNNTLY SEQ ID NO: 66 GDDGNNTLI SEQ ID NO: 504 GDSGDNTLV SEQ ID NO: 942
  • GGGGNNTLY SEQ ID NO: 67 GGAGDNTLI SEQ ID NO: 505 GGGGDNTLV SEQ ID NO: 943
  • GNGGNNTLY SEQ ID NO: 68 GNAGDNTLI SEQ ID NO: 506 GNGGDNTLV SEQ ID NO: 944
  • GDGGNNTLY SEQ ID NO: 69
  • GDAGDNTLI SEQ ID NO: 507
  • GDGGDNTLV SEQ ID NO: 945
  • GNDGNNTLY SEQ ID NO: 71 GNSGDNTLI SEQ ID NO: 509 GNDGDNTLV SEQ ID NO: 947
  • GDDGNNTLY SEQ ID NO: 72
  • GDSGDNTLI SEQ ID NO: 510
  • GDDGDNTLV SEQ ID NO: 948
  • GGAGDNTLY SEQ ID NO: 73
  • GGGGDNTLI SEQ ID NO: 511
  • GGAGANTLV SEQ ID NO: 949
  • GNAGDNTLY SEQ ID NO: 74
  • GNGGDNTLI SEQ ID NO: 512 GNAGANTLV SEQ ID NO: 950
  • GDAGDNTLY SEQ ID NO: 75
  • GDGGDNTLI SEQ ID NO: 513 GDAGANTLV SEQ ID NO: 951 Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO)
  • GGSGDNTLY SEQ ID NO 76
  • GGDGDNTLI SEQ ID NO: 514
  • GGSGANTLV SEQ ID NO: 952
  • GNSGDNTLY SEQ ID NO 77
  • GNDGDNTLI SEQ ID NO: 515
  • GNSGANTLV SEQ ID NO: 953
  • GDSGDNTLY SEQ ID NO 78
  • GDDGDNTLI SEQ ID NO: 516
  • GDSGANTLV SEQ ID NO: 954
  • GGGGDNTLY SEQ ID NO 79 GGAGANTLI SEQ ID NO: 517 GGGGANTLV SEQ ID NO: 955
  • GNGGDNTLY SEQ ID NO 80 GNAGANTLI SEQ ID NO: 518 GNGGANTLV SEQ ID NO: 956
  • GDGGDNTLY SEQ ID NO 81 GDAGANTLI SEQ ID NO: 519 GDGGANTLV SEQ ID NO: 957
  • GGDGDNTLY SEQ ID NO 82 GGSGANTLI SEQ ID NO: 520 GGDGANTLV SEQ ID NO: 958
  • GNDGDNTLY SEQ ID NO 83 GNSGANTLI SEQ ID NO: 521 GNDGANTLV SEQ ID NO: 959
  • GDDGDNTLY SEQ ID NO 84 GDSGANTLI SEQ ID NO: 522 GDDGANTLV SEQ ID NO: 960
  • GGAGANTLY SEQ ID NO 85 GGGGANTLI SEQ ID NO: 523 GGAGNDILV SEQ ID NO: 961
  • GNAGANTLY SEQ ID NO 86 GNGGANTLI SEQ ID NO: 524 GNAGNDILV SEQ ID NO: 962
  • GDAGANTLY SEQ ID NO 87 GDGGANTLI SEQ ID NO: 525 GDAGNDILV SEQ ID NO: 963
  • GDSGANTLY SEQ ID NO 90 GDDGANTLI SEQ ID NO: 528 GDSGNDILV SEQ ID NO: 966
  • GNDGANTLY SEQ ID NO 95 GNSGNDILI SEQ ID NO: 533 GNDGNDILV SEQ ID NO: 971
  • GNAGNDILY SEQ ID NO 98 GNGGNDILI SEQ ID NO: 536 GNAGDDILV SEQ ID NO: 974
  • GDAGNDILY SEQ ID NO 99 GDGGNDILI SEQ ID NO: 537 GDAGDDILV SEQ ID NO: 975
  • GGSGNDILY SEQ ID NO 100 GGDGNDILI SEQ ID NO: 538 GGSGDDILV SEQ ID NO: 976
  • GNSGNDILY SEQ ID NO 101 GNDGNDILI SEQ ID NO: 539 GNSGDDILV SEQ ID NO: 977
  • GDSGNDILY SEQ ID NO 102 GDDGNDILI SEQ ID NO: 540 GDSGDDILV SEQ ID NO: 978
  • GDGGNDILY SEQ ID NO 105 GDAGDDILI SEQ ID NO: 543 GDGGDDILV SEQ ID NO: 981
  • GGDGNDILY SEQ ID NO 106 GGSGDDILI SEQ ID NO: 544 GGDGDDILV SEQ ID NO: 982
  • GNDGNDILY SEQ ID NO 107 GNSGDDILI SEQ ID NO: 545 GNDGDDILV SEQ ID NO: 983
  • GDDGNDILY SEQ ID NO 108 GDSGDDILI SEQ ID NO: 546 GDDGDDILV SEQ ID NO: 984
  • GNAGDDILY SEQ ID NO 110 GNGGDDILI SEQ ID NO: 548 GNAGADILV SEQ ID NO: 986
  • GDAGDDILY SEQ ID NO 111 GDGGDDILI SEQ ID NO: 549 GDAGADILV SEQ ID NO: 987
  • GDSGDDILY SEQ ID NO 114 GDDGDDILI SEQ ID NO: 552 GDSGADILV SEQ ID NO: 990
  • GDDGDDILY SEQ ID NO 120 GDSGADILI SEQ ID NO: 558 GDDGADILV SEQ ID NO: 996
  • GDAGADILY SEQ ID NO 123 GDGGADILI SEQ ID NO: 561 GDAGNNILV SEQ ID NO: 999
  • GNDGADILY SEQ ID NO 131 GNSGNNILI SEQ ID NO 569 GNDGNNILV SEQ ID NO 1007
  • GNDG NILY SEQ ID NO 143 GNSGDNILI SEQ ID NO 581 GNDGDNILV SEQ ID NO 1019
  • GDDG NILY SEQ ID NO 144 GDSGDNILI SEQ ID NO 582 GDDGDNILV SEQ ID NO 1020
  • GGAGDNILY SEQ ID NO 145 GGGGDNILI SEQ ID NO 583 GGAGANILV SEQ ID NO 1021
  • GNAGDNILY SEQ ID NO 146 GNGGDNILI SEQ ID NO 584 GNAGANILV SEQ ID NO 1022
  • GDAGDNILY SEQ ID NO 147
  • GDGGDNILI SEQ ID NO 585
  • GDAGANILV SEQ ID NO 1023
  • GGSGDNILY SEQ ID NO 148
  • GGDGDNILI SEQ ID NO 586
  • GGSGANILV SEQ ID NO 1024
  • GNSGDNILY SEQ ID NO 149 GNDGDNILI SEQ ID NO 587 GNSGANILV SEQ ID NO 1025
  • GDGGDNILY SEQ ID NO 153 GDAGANILI SEQ ID NO 591 GDGGANILV SEQ ID NO 1029
  • GGDGDNILY SEQ ID NO 154 GGSGANILI SEQ ID NO 592 GGDGANILV SEQ ID NO 1030
  • GNDGANILY SEQ ID NO 167 GNSGNDVLI SEQ ID NO 605 GNDGNDVLV SEQ ID NO 1043
  • GGAGNDVLY SEQ ID NO 169 GGGGNDVLI SEQ ID NO 607 GGAGDDVLV SEQ ID NO 1045
  • GGSGNDVLY SEQ ID NO 172
  • GGDGNDVLI SEQ ID NO 610
  • GGSGDDVLV SEQ ID NO 1048
  • GGSGDDVLY SEQ ID NO: 184
  • GGDGDDVLI SEQ ID NO 622
  • GGSGADVLV SEQ ID NO 1060
  • GNAG VLY SEQ ID NO: 206 GNGGNNVLI SEQ ID NO 644 GNAGDNVLV SEQ ID NO 1082
  • GGSG NVLY SEQ ID NO: 208 GGDGNNVLI SEQ ID NO 646 GGSGDNVLV SEQ ID NO 1084
  • GNSG NVLY SEQ ID NO: 209
  • GNDGNNVLI SEQ ID NO 647
  • GNSGDNVLV SEQ ID NO 1085
  • GDSG NVLY SEQ ID NO: 210 GDDGNNVLI SEQ ID NO 648 GDSGDNVLV SEQ ID NO 1086
  • GGDG VLY SEQ ID NO: 214 GGSGDNVLI SEQ ID NO 652 GGDGDNVLV SEQ ID NO 1090
  • GDDG VLY SEQ ID NO: 216 GDSGDNVLI SEQ ID NO 654 GDDGDNVLV SEQ ID NO 1092
  • GGAGD VLY SEQ ID NO: 217 GGGGDNVLI SEQ ID NO 655 GGAGANVLV SEQ ID NO 1093
  • GNAGD VLY SEQ ID NO: 218 GNGGDNVLI SEQ ID NO 656 GNAGANVLV SEQ ID NO 1094
  • GDAGD VLY SEQ ID NO: 219 GDGGDNVLI SEQ ID NO 657 GDAGANVLV SEQ ID NO 1095
  • GGSGDNVLY SEQ ID NO: 220
  • GGDGDNVLI SEQ ID NO 658
  • GGSGANVLV SEQ ID NO 1096
  • GNSGDNVLY SEQ ID NO: 221 GNDGDNVLI SEQ ID NO 659 GNSGANVLV SEQ ID NO 1097
  • GDSGDNVLY SEQ ID NO: 222 GDDGDNVLI SEQ ID NO 660 GDSGANVLV SEQ ID NO 1098
  • GDGGD VLY SEQ ID NO: 225 GDAGANVLI SEQ ID NO 663 GDGGANVLV SEQ ID NO 1101
  • GGDGD VLY SEQ ID NO: 226 GGSGANVLI SEQ ID NO 664 GGDGANVLV SEQ ID NO 1102
  • GDAGA VLY SEQ ID NO: 231 GDGGANVLI SEQ ID NO 669 GDAGNDTIV SEQ ID NO 1107
  • GGSGANVLY SEQ ID NO: 232
  • GGDGANVLI SEQ ID NO 670
  • GGSGNDTIV SEQ ID NO 1108
  • GNSGANVLY SEQ ID NO: 233 GNDGANVLI SEQ ID NO 671 GNSGNDTIV SEQ ID NO 1109
  • GDSGANVLY SEQ ID NO: 234 GDDGANVLI SEQ ID NO 672 GDSGNDTIV SEQ ID NO 1110 Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO)
  • GGGGANVLY SEQ ID NO 235 GGAGNDTII SEQ ID NO: 673
  • GGGGNDTIV SEQ ID NO 1111
  • GNGGANVLY SEQ ID NO 236 GNAGNDTII SEQ ID NO: 674 GNGGNDTIV SEQ ID NO 1112
  • GDGGANVLY SEQ ID NO 237 GDAGNDTII SEQ ID NO: 675 GDGGNDTIV SEQ ID NO 1113
  • GGDGANVLY SEQ ID NO 238 GGSGNDTII SEQ ID NO: 676 GGDGNDTIV SEQ ID NO 1114
  • GNDGANVLY SEQ ID NO 239 GNSGNDTII SEQ ID NO: 677 GNDGNDTIV SEQ ID NO 1115
  • GDDGANVLY SEQ ID NO 240 GDSGNDTII SEQ ID NO: 678 GDDGNDTIV SEQ ID NO 1116
  • GDSGNNTIY SEQ ID NO 282 GDDGNNTII SEQ ID NO: 720 GDSGDNTIV SEQ ID NO 1158
  • GDAGDNTIY SEQ ID NO 291
  • GDGGDNTII SEQ ID NO: 729
  • GDAGANTIV SEQ ID NO 1167
  • GDDGDNTIY SEQ ID NO 300 GDSGANTII SEQ ID NO: 738 GDDGANTIV SEQ ID NO 1176
  • GGDGDNIIY SEQ ID NO 370 GGSGANIII SEQ ID NO: 808 GGDGANIIV SEQ ID NO 1246
  • GDGGA VIY SEQ ID NO: 453 GDAGNDTLV SEQ ID NO: 891 GDNASDLFN SEQ ID NO 1329
  • GNAGNDTLI SEQ ID NO: 458 GNGGNDTLV SEQ ID NO: 896 GDLASDLFK SEQ ID NO 1334
  • GDAGNDTLI SEQ ID NO: 459
  • GDGGNDTLV SEQ ID NO: 897
  • GDNASDLFK SEQ ID NO 1335
  • GGSGNDTLI SEQ ID NO: 460
  • GGDGNDTLV SEQ ID NO: 898
  • GDEASDLFS SEQ ID NO 1336
  • GNSGNDTLI SEQ ID NO: 461
  • GNDGNDTLV SEQ ID NO: 899
  • GDLASDLFS SEQ ID NO 1337
  • GDSGNDTLI SEQ ID NO: 462 GDDGNDTLV SEQ ID NO: 900
  • PBRC precipatable-beta roll cassette
  • a PBRC will comprise at least two PBRTs.
  • a PBRC will comprise at least 3 PBRTs, at least 4 PBRTs, at least 5 PBRTs, at least 6 PBRTs, at least 7 PBRTs, at least 8 PBRTs, at least 9 PBRTs, at least 10 PBRTs, at least 11 PBRTs, at least 12 PBRTs, at least 13 PBRTs, at least 14 PBRTs, at least 15 PBRTs, at least 16 PBRTs, at least 17 PBRTs, at least 18 PBRTs, at least 19 PBRTs, at least 20 PBRTs, or 20 or more PBRTs.
  • the PBRC will comprise at least two PBRTs.
  • a PBRC will comprise at least 3 PBRTs, at least 4 PBRTs, at least 5 PBRTs, at least 6 PBRTs, at least 7 PB
  • PBRCs described herein will comprise a plurality of precipatable beta roll tags arranged in a tandem repeat.
  • the PBRCs described herein can
  • PBRTs comprise at least 2 PBRTs, at least 3 PBRTs, at least 4 PBRTs, at least 5 PBRTs, at least 6
  • a PBRC can comprise at least two PBRCs separated by a linking amino acid sequence.
  • a linking amino acid sequence in present between two PBRTs a PBRT located at either end of the linking sequence can be an individual PBRT or it can be a PBRT that is part of a tandem arrangement of two or more
  • the PBRCs can comprise polymeric or oligomeric repeats of a PBRT.
  • PBRT polymeric or oligomeric repeats of a PBRT.
  • the PBRCs described herein can comprise one or more different PBRTs.
  • all of the PBRTs comprised in a PBRC are identical in amino acid sequence.
  • all of the PBRTs comprised in a PBRC have different amino acid sequences.
  • at least one PBRT comprised in a PBRC has a different amino acid sequence as compared to another PBRT in the PBRC.
  • the PBRCs described herein can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 different PBRTs.
  • a PBRC can also comprise a capping sequence (“CS") refers to the an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 3.
  • CS capping sequence
  • capping sequence also refers to a capping sequence having the amino acid sequence of any of SEQ ID NO: 4-23.
  • polypeptide comprising one or more PBRT to undergo reversible Ca2+
  • the capping sequence is an amino acid sequence, which, when located C-terminally or N- terminally to one or more PBRTs, allows the one or more PBRTs bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the PBRC.
  • a PBRC will comprise at least 2 PBRTs, at least 3 PBRTs, at least 4 PBRTs, at least 5 PBRTs, at least 6 PBRTs, at least 7 PBRTs, at least 8 PBRTs, at least 9 PBRTs, at least 10 PBRTs, at least 11 PBRTs, at least 12 PBRTs, at least 13 PBRTs, at least 14 PBRTs, at least 15 PBRTs, at least 16 PBRTs, at least 17 PBRTs, at least 18 PBRTs, at least 19 PBRTs, at least 20 PBRTs, or 20 or more PBRTs, all of which are located N-terminally to a CS.
  • a PBRC will comprise 2, at least 3 PBRTs, at least 4 PBRTs, at least 5 PBRTs, at least 6 PBRTs, at least 7 PBRTs, at least 8 PBRTs, at least 9 PBRTs, at least 10 PBRTs, at least 11 PBRTs, at least 12 PBRTs, at least 13 PBRTs, at least 14 PBRTs, at least 15 PBRTs, at least 16 PBRTs, at least 17 PBRTs, at least 18 PBRTs, at least 19 PBRTs, at least 20 PBRTs, or 20 or more PBRTs, all of which are located C-terminally to a CS.
  • a PBRC is an amino acid sequence comprising at least five tandem PBRTs situated N-terminally to a capping sequence.
  • a PBRC is an amino acid sequence comprising at least six to about 16 tandem PBRTs situated N-terminally to a capping sequence.
  • a precipatable beta roll cassette is an amino acid sequence comprising 17 or more tandem PBRTs situated N-terminally to a capping sequence.
  • the capping sequence comprises the sequence
  • INAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQAGI EKLVEAMAQYPD (SEQ ID NO: 3) which is a C-terminal sequence on the block V beta roll domain of the adenylate cyclase toxin of B. pertussis.
  • a PBRC comprises the amino acid sequence
  • a PBRC comprises the amino acid sequence
  • the capping sequence comprises the sequence
  • the capping sequence comprises the sequence INAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEAIHAANQAIDPAGI EKLVEAMAQYPD (SEQ ID NO: 7) (adenylate cyclase-hemolysin [Bordetella
  • the capping sequence comprises the sequence INAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEAIHAANQTVDPAGI EKLVEAMAQYPD (SEQ ID NO: 8) (adenylate cyclase hemolysin [Bordetella
  • the capping sequence comprises the sequence QLWFSKSGSDLEVRVVGTDDAVTVAGWYSGAEHHMDSIETADGTVLLDSMVDRLV QAMAGF (SEQ ID NO: 9) (Azospirillum sp. B510 calcium binding hemolysin protein).
  • the capping sequence comprises the sequence ADQLWFRHVGNDLEISILGTGDTATVRDWYLGSRYQIEQIRVDDGRTLVNADVEKL VQAMA (SEQ ID NO: 10) (hemolysin-type calcium-binding region Burkholderia cenocepacia MCO-3).
  • the capping sequence comprises the sequence ADQLWFRHVGNDLEISILGSSDTATVRDWYSGSRYQIEQIRLDDGRTLVNADVEKLV QAM A (SEQ ID NO: 11) (hemolysin-type calcium-binding region [Burkholderia ambifaria MC40-6]). In another embodiment, the capping sequence comprises the sequence
  • the capping sequence comprises the sequence
  • DARQTNLWFSQVGKDLQIDVLGSTDQVTVKDWYAGADNRVERIKTADGKTLYDSD VDKLVQAMASF (SEQ ID NO: 13) (calcium binding secreted hemolysin protein
  • the capping sequence comprises the sequence
  • the capping sequence comprises the sequence
  • EELWFSRDGNDLQINVIGTDNQVEISDWYSGVNYQLDKVQVGDSVLLNTQLEQLVS AMASF SEQ ID NO: 15 (hemolysin-type calcium binding protein [Shewanella
  • the capping sequence comprises the sequence GLSELWFSRENNDLIIKSLLSEDKVTVQNWYSHQDHKIENIRLSNEQMLVSTQVEKM VESMAGF (SEQ ID NO: 16) (RTX toxin protein [Actinobacillus pleuropneumoniae serovar 10). In another embodiment, the capping sequence comprises the sequence
  • the capping sequence comprises the sequence
  • the capping sequence comprises the sequence
  • the capping sequence comprises the sequence
  • the capping sequence comprises the sequence
  • the capping sequence comprises the sequence
  • the capping sequence comprises the sequence
  • polypeptide comprising one or more PBRT to undergo reversible Ca2+
  • the one or more PBRTs can require that the one or more PBRTs be located N-terminally or C- terminally to a stabilizing polypeptide. It is known that when a certain stabilizing
  • polypeptides e.g. GFP, maltose binding protein
  • GFP GFP, maltose binding protein
  • a PBRC is an amino acid sequence comprising one or more PBRTs located N-terminally or C-terminally to a stabilizing polypeptide, wherein the stabilizing polypeptide cane be, but is not limited to, glutathione S-transferase (GST), maltose E binding protein (MBP), Green Fluorescent Protein (GFP), and variants thereof.
  • GST glutathione S-transferase
  • MBP maltose E binding protein
  • GFP Green Fluorescent Protein
  • the stabilizing polypeptide is an amino acid sequence of any amino acid composition wherein the sequence comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least
  • the PBRCs described herein can further comprise one or more cleavage sites.
  • the cleavage site can be positioned C- terminally or N-terminally to a PBRC so as to allow for cleavage of a PBRC from a linked purification moiety (e.g. a polypeptide purification moiety linked to a PBRC as part of a fusion protein).
  • a first cleavage site can be positioned C-terminally or N-terminally to a PBRC so as to allow for cleavage of a PBRC from a linked purification moiety (e.g.
  • a polypeptide purification moiety linked to a PBRC as part of a fusion protein and a second cleavage site can be positioned between a PBRT and a capping sequence or a PBRT and a stabilizing polypeptide so as to allow so as to allow for cleavage of the capping sequence or the stabilizing polypeptide from the one or more PBRTs in the PBRC.
  • cleavage at such cleavage sites can be useful for purification of a purification moiety of interest.
  • the cleavage site is a proteolytic cleavage site.
  • exemplary proteolytic cleavage sites include, but are not limited to Factor Xa, thrombin, or
  • the cleavage site is a signal peptidase cleavage site.
  • the cleavage site is a self cleaving intein cleavage site (Amitai et al., Proceedings of the National Academy of Sciences, vol. 106, no. 27, pp. 11005 -11010, Jul. 2009; Hiraga et al., Journal of Molecular Biology, vol. 393, no. 5, pp. 1106-1117, Nov. 2009). Any other specific cleavage sites known in the art can be used in connection with the methods described herein.
  • the PBRTs or PBRCs described herein may be linked to a purification moiety by any means known in the art.
  • the PBRTs or PBRCs described herein ca be located at any site in a polypeptide comprising a purification moiety of interest, including a location that is N- terminal, a location that is C-terminal or a location within the sequence of the purification moiety of interest.
  • the PBRTs or PBRCs described herein can also be chemically linked to purification moieties other than by means of a fusion protein.
  • reference to a PBRC linked purification moiety, or to a PBRT linked purification moiety encompasses for purification moieties linked to a PBRC or PBRT by peptide linkage (e.g. as a fusion protein) or by non-peptide bond chemical linkage.
  • the chemical modification of PBRTs or PBRCs described herein can be performed according to any method known in the art..
  • amides of the PBRTs or PBRCs described herein can be prepared by techniques well known in the art for converting a carboxylic acid group or precursor, to an amide.
  • One method for amide formation at the C- terminal carboxyl group is to cleave the polypeptide, or fusion thereof from a solid support with an appropriate amine, or to cleave in the presence of an alcohol, yielding an ester, followed by aminolysis with the desired amine.
  • Salts of carboxyl groups of the PBRTs or PBRCs described herein can be prepared by contacting the polypeptide, or fusion thereof with one or more equivalents of a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide; a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate; or an amine base such as, for example, triethylamine, triethanolamine, and the like.
  • a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide
  • a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate
  • an amine base such as, for example, triethylamine, triethanolamine, and the like.
  • N-acyl derivatives of an amino group of the PBRTs or PBRCs described herein can be prepared by utilizing an N-acyl protected amino acid for the final condensation, or by acylating a protected or unprotected polypeptide, or fusion thereof.
  • O-acyl derivatives can be prepared, for example, by acylation of a free hydroxy polypeptide or polypeptide resin. Either acylation can be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N- and O-acylation can be carried out together, if desired.
  • Formyl-methionine, pyroglutamine and trimethyl-alanine can be substituted at the N- terminal residue of PBRTs or PBRCs described herein.
  • Other amino-terminal modifications include aminooxypentane modifications.
  • Such chemical linkages can be useful for purifying non-peptide molecules such as lipids, oligonucleotides and carbohydrates, small organic or inorganic molecules, proteins, single-stranded or double-stranded oligonucleotides, polynucleotides, metals (e.g. cobalt, zinc, nickel or copper) and the like.
  • the chemically modified PBRTs or PBRCs described herein can be assayed for the ability to bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the PBRT or PBRC using methods known to those skilled in the art.
  • the PBRTs and PBRCs described herein can also be coupled with a radioisotope or enzymatic label to facilitate their detection.
  • the PBRTs or PBRCs described herein can be isotopically-labeled where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring).
  • Suitable radionuclides that may be incorporated in compounds of the present invention include but are not limited to 2 H (also written as D for deuterium), 3 H (also written as T for tritium), n C, 13 C, 14 C, 13 N, 15 N, 15 0, 17 0, 18 0, 18 F, 35 S, 36 C1, 82 Br, 75 Br, 76 Br, 77 Br, 123 1, 124 I, 125 I and 131 I.
  • the radionuclide that is incorporated in the instant radio-labeled compounds can depend on the specific application of that radio-labeled compound.
  • the PBRTs or PBRCs described herein can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the PBRTs or PBRCs described herein can be labeled with a fluorescent dye, spin label, heavy metal or radio-labeled peptides.
  • Esters of carboxyl groups of the PBRTs or PBRCs described herein can also be prepared by any of the usual methods known in the art.
  • the methods and compositions described herein are useful in a broad range of bioseparation applications.
  • the methods and compositions described herein can be used for rapid expression and purification of a purification moiety linked to a PBRC.
  • PBRC linked purification moieties can be expressed in any number of expression systems, including in vitro and in vivo expression systems.
  • Exemplary in vivo expression systems suitable for expressing the PBRC linked purification moieties described herein include, but are not limited to, bacterial systems, yeast systems, and mammalian systems.
  • the invention relates to a method for purifying purification moieties (e.g. a PBRC or a purification moiety linked to a PBRC).
  • purification moieties e.g. a PBRC or a purification moiety linked to a PBRC.
  • the methods described herein can be used for purifying one or more purification moieties from a heterogeneous mixture of biomaterials in a sample.
  • the methods descried herein can be used to purify chemically synthesized purification moieties or in-vitro synthesized purification moieties.
  • the bioseparation methods described herein can comprise expressing a PBRT linked purification moiety (e.g. a PBRC fusion protein) in a cellular expression system (e.g. a bacterial cell).
  • a PBRC linked purification moiety e.g. a PBRC fusion protein
  • the PBRC linked purification moiety can then be released into a medium by cell lysis. Any method of cell lysis known in the art can be used in conjunction with the methods described herein, including, but not limited to chemical lysis (e.g. detergents) or physical methods (e.g. sonication or French press).
  • the PBRC or PBRC linked purification moiety can be expressed in an in-vitro expression system (e.g. a rabbit reticulocyte system) such that the purification moiety is expressed into the expression system medium.
  • bioseparation can be achieved by increasing the free Ca2+ concentration in the medium comprising the PBRC linked purification moiety to induce precipitation of the PBRC linked purification moiety, followed by removing material that does not precipitate from the medium and then resuspending the precipitated material in a medium having a reduced Ca2+ concentration or in a medium having a reduced concentration of free Ca2+ (e.g. a medium comprising a Ca2+ chelators such as EDTA). These steps can be repeated until the desired level of purity is reached.
  • the PBRC linked purification moiety can be expressed in a cellular expression system, and bioseparation can be achieved by increasing the free Ca2+ concentration within the cell prior to cellular lysis.
  • Many methods for increasing intracellular Ca2+ concentrations are known in the art, including, but not limited to adding Ca2+ to a cellular medium, with or without presence of ionophores or cell permeabilization agents.
  • the cells can then be subjected to lysis conditions (e.g. chemical lysis or physical lysis) and the resulting precipitate can be recovered.
  • Precipitated PBRC or the PBRC linked purification moieties can then be recovered by reducing the free Ca2+ concentration (e.g.
  • the PBRC linked purification moieties described herein can further comprise a peptide sequence to induce secretion into the periplasm of a cell (e.g. an E. coli cell) or to the medium outside of a cell.
  • a cell e.g. an E. coli cell
  • cell lysis may be required for further purification of the PBRC linked purification moiety.
  • purification can be achieved without cell lysis by eliminating intact cells (e.g. by centrifugation) and purification of the PBRC linked purification moiety from the extracellular medium by increasing the free Ca2+ concentration of the supernatant.
  • the adjustment of conditions during the purification process can be achieved by numerous methods, including, but not limited to, adjusting the temperature, pH or salt concentration of the aqueous media.
  • a purified PBRT linked purification moiety can contain less than about 50%, less than about 75%, or less than about 90%, of the materials with which it was originally associated.
  • a purification moiety of interest can be linked to PBRC comprising a cleavable peptide sequence (e.g. a self-cleaving peptide sequence such an intein) positioned between the PBRC and the purification moiety.
  • a cleavable peptide sequence e.g. a self-cleaving peptide sequence such an intein
  • the PBRC linked purification moiety can be recovered using standard techniques as either a homogenous mixture or as a heterogeneous sample. The mixture can then be exposed to calcium to induce precipitation of the PBRC linked purification moiety.
  • the PBRC linked purification moiety can then be resuspended in buffer that has reduced Ca2+, that has reduced free Ca2+ or that contains a calcium chelator (e.g. EDTA).
  • the PBRC linked purification moiety can then ne subjected condition that cause cleavage to separate the PBRC from the purification moiety and calcium can be once again added to the mixture. This will precipitate out the PBRC moiety and thereby leaving behind a sample of purified purification moiety of interest.
  • PBRC linked purification moiety can be a moiety which binds a second molecule and the second molecule can be used to remove the purification moiety from the sample (e.g. a resin or beads coated with the second molecule) after induced cleavage at a site between the purification moiety and the PBRC.
  • immobilization of the PBRC linked purification moieties described herein or its binding proteins can be used to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.
  • Immobilization of the PBRC linked purification moieties described herein can be by linking to a solid support, including a plastic or glass plate or bead, a chromatographic resin, a filter or a membrane. Methods of attachment of proteins, or membranes containing same, to such supports are well known in the art. Immobilization of the PBRC linked purification moieties described herein can also be accomplished in any vessel suitable for containing the reactants.
  • a fusion protein can be provided which adds a domain that allows the PBRT linked purification moiety described herein to be bound to a matrix.
  • glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads or glutathione derivatized microtiter plates, which are then combined with the cell lysates, and the mixture incubated under conditions conducive to complex formation.. Following incubation, the beads can be washed to remove any unbound fraction,. Alternatively, the complexes can be dissociated from the matrix using standard electrophoretic techniques.
  • PBRCs undergo a reversible Ca2+ binding dependent transition.
  • PBRCs or PBRC linked purification moieties undergo reversible precipitation at a Ca2+ concentration (or free Ca2+) phase transition concentration.
  • transition concentrations reversible and the isolated precipatable beta-roll tags or purification moieties comprising a precipatable beta-roll tag can be completely resolubilized in a medium below a certain Ca2+ concentration (or free Ca2+) transition concentration, through, for example the addition of a calcium chelator into the medium comprising the PBRC or PBRC linked purification moiety.
  • the concentration of Ca2+ required to induce reversible precipitation of a PBRC or PBRC linked purification moiety can be readily determined by adding increasing amounts of Ca2+ until such time as the PBRC or PBRC linked purification moiety begins to precipitate from a the medium. One can readily determine the extent of precipitation by centrifuging the medium.
  • the amount of Ca2+ required to induce reversible precipitation a PBRC of PBRC linked purification moiety will be about lmM Ca2+, more than about ImM Ca2+, more than about 5mM Ca2+, more than about lOmM Ca2+, more than about 20mM Ca2+, more than about 30mM Ca2+, more than about 50mM Ca2+, more than about 75mM Ca2+, more than about 100 mM Ca2+, more than about 150mM Ca2+, more than about 200mM Ca2+, or more than about 500mM Ca2+.
  • the amount of Ca2+ required to induce precipitation of a PBRC or a PBRC linked purification moiety can increase as a function of the number of PBRTs in the PBRC.
  • a PBRC linked purification moiety comprising 8 PBRTs may precipitate in 150mM Ca2+ wherein a PBRC linked purification moiety comprising 17 PBRTs may precipitate in 25mM Ca2+.
  • One of skill in the art will readily be capable of determining the amount of Ca2+ required to precipitate a particular PBRC or a particular PBRC linked purification moiety simply by titrating increasing concentrations of Ca2+.
  • the concentration of Ca2+ required to reverse precipitation of a PBRT or PBRC or of a PBRT or PBRC linked purification moiety can be readily determined by reducing the concentration of free Ca2+ in a medium until such time as a precipitated PBRC or PBRC linked purification moiety begins to solubilize into the medium.
  • concentration of Ca2+ required to reverse precipitation of a PBRT or PBRC or of a PBRT or PBRC linked purification moiety can be readily determined by reducing the concentration of free Ca2+ in a medium until such time as a precipitated PBRC or PBRC linked purification moiety begins to solubilize into the medium.
  • One can readily determine the extent of precipitation by centrifuging the medium.
  • the amount of free Ca2+ in the medium required to solubilize a precipitated PBRT or PBRC or of a PBRT or PBRC linked purification moiety will be less than about ImM Ca2+, less than about ImM Ca2+, less than about 5mM Ca2+, less than about lOmM Ca2+, less than about 20mM Ca2+, less than about 30mM Ca2+, less than about 50mM Ca2+, less than about 75mM Ca2+, less than about 100 mM Ca2+, less than about 150mM Ca2+, less than about 200mM Ca2+, or less than about 500mM Ca2+.
  • the free Ca2+ concentration required to reverse precipitation of a PBRC or a PBRC linked purification moiety can correlated to the number of PBRTs in the PBRC.
  • a PBRC linked purification moiety comprising 8 PBRTs may become soluble in a higher free Ca2+ concentration as compared to a PBRC linked purification moiety comprising 17 PBRTs.
  • One of skill in the art will readily be capable of determining the Ca2+ concentration required to reverse precipitation a particular PBRC or a particular PBRC linked purification moiety simply by decreasing free Ca2+ concentrations.
  • the free Ca2+ concentration of a medium comprising a PBRC or a PBRC linked purification moiety can be reduced by adding one or more calcium chelators into the medium.
  • Any number of calcium chelators can be used in the connection with the methods described herein. Examples of suitable calcium chelators include, but are not limited to EDTA, EGTA, and BAPTA.
  • the amount of a calcium chelator required to solubilize a precipitated PBRT or PBRC or of a PBRT or PBRC linked purification moiety will be about ImM Ca2+, more than about ImM Ca2+, more than about 5mM Ca2+, more than about lOmM Ca2+, more than about 20mM Ca2+, more than about 30mM Ca2+, more than about 50mM Ca2+, more than about 75mM Ca2+, more than about 100 mM Ca2+, more than about 150mM Ca2+, more than about 200mM Ca2+, or more than about 500mM Ca2+.
  • PBRT or PBRC linked purification moieties described herein include pH, the addition of organic solutes and solvents, side-chain ionization or chemical modification, and pressure.
  • PBRC linked purification moieties described herein can be further purified or isolated according to any method of protein purification or isolation known in the art.
  • PBRCs or PBRC linked purification moieties can be purified by various methods including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, precipitation and salting-out chromatography, extraction, and countercurrent distribution.
  • the PBRCs or the PBRC linked purification moieties can be produced in a recombinant system in which the protein contains an additional sequence tag that facilitates purification, such as, but not limited to, a polyhistidine sequence, or a sequence that specifically binds to an antibody, such as FLAG and GST.
  • the PBRCs or the PBRC linked purification moieties can be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix.
  • antibodies produced against the PBRTs or PBRCs, or a PBRC linked purification moiety or against polypeptides derived therefrom can be used as purification reagents.
  • the methods and compositions described herein can be useful for the detection of a broad range of purification moieties in biosensing applications.
  • the methods and compositions described herein can be used for the separation of protein of interest from a sample for detection of bimolecular interactions.
  • the PBRC linked antibody can be added to a sample.
  • Ca2+ can then be added to the sample to induce precipitation of the antibody such that antigen that interact with, or form a complex with, the antibody also precipitate upon the addition of Ca2+.
  • the precipitate can then be collected and resuspended and the sample can be characterized.
  • PBRC PBRC-derived polypeptide capable of binding to a second purification moiety
  • Ca2+ can then be added to the sample to induce precipitation of the polypeptide such that other purification moieties that interact with, or form a complex with, the polypeptide also precipitate upon the addition of Ca2+.
  • the precipitate can then be collected and resuspended and the sample can be characterized.
  • the presence and quantity of the target in the original sample, as well as any associated additional purification moieties, can be characterized and determined.
  • the PBRC linked purification moieties described herein can be produced in prokaryotic or eukaryotic host cells by expression of nucleic acids encoding a polypeptide of this invention. The production of these polypeptides can also be done as part of a larger polypeptide.
  • the PBRC linked purification moieties described herein can also be synthesized in vitro, e.g., by the solid phase polypeptide synthetic method or by recombinant DNA approaches described herein.
  • the solid phase polypeptide synthetic method is an established and widely used method.
  • These PBRC or PBRC linked purification moieties described herein can be further purified by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on an anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; or ligand affinity chromatography.
  • PBRC linked purification moieties described herein can also be produced using any in-vitro expression system known in the art or can be synthesized by chemical methods. Methods for expression of heterologous proteins in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in the art and are described further in Sambrook J et al.; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif; Gutte B and Merrifield R B, J. Am. Chem. Soc. 91 :501-02 (1969); Chaiken I M, CRC Crit. Rev. Biochem.
  • Exemplary peptide synthesis methods known in the art include, but are not limited to those described in Stewart et al., Solid Phase Peptide Synthesis, Pierce Biotechnology, Inc., Rockford, 111., 1984; Bodanszky, Principles of Peptide Synthesis, Springer-Verlag, New York, 1984; and Pennington et al., Peptide Synthesis Protocols, Humana Press, Totowa, N.J., 1994). Additionally, many companies offer custom peptide synthesis services.
  • the PBRC linked purification moieties described herein can also be produced by direct chemical synthesis.
  • the PBRC linked purification moieties described herein can be produced as modified polypeptides, with nonpeptide moieties attached by covalent linkage to the N-terminus and/or C-terminus.
  • either the carboxy-terminus or the amino-terminus, or both are chemically modified. Common modifications of the terminal amino and carboxyl groups, include, but are not limited to acetylation and amidation, respectively.
  • Amino-terminal modifications such as acylation (e.g., acetylation) or alkylation (e.g., methylation) and carboxy-terminal-modifications such as amidation, as well as other terminal modifications, including cyclization, can be incorporated into various embodiments.
  • Certain amino-terminal and/or carboxy-terminal modifications and/or polypeptide extensions to the core sequence can provide advantageous physical, chemical, biochemical, and pharmacological properties, such as: enhanced stability, increased potency and/or efficacy, resistance to serum proteases, desirable pharmacokinetic properties, and others.
  • the PBRC linked purification moieties can be prepared using recombinant DNA and molecular cloning techniques. Genes encoding the PBRC linked purification moieties may be produced in heterologous host cells, particularly in the cells of microbial hosts. Any techniques for transfecting host cells and purifying proteins and polypeptides known in the art can be used in connection with the methods described herein. Exemplary epitope tags suitable for use with the methods described herein include, but are not limited to FLAG, HA, Myc and T7 epitope tags.
  • the PBRTs, PBRCs or PBRC linked purification moieties described herein can be synthesized chemically using standard polypeptide synthesis techniques.
  • the invention also extends to the DNA expression vector comprising DNA coding for the PBRTs or PBRCs described herein, whether or not the encoded products further comprise a linked purification moiety.
  • the invention also provides the expression vector comprising sequences coding for a PBRT or a PBRC configured to allow insertion of a DNA sequence downstream of the sequence coding for the PBRT or the PBRC so as to facilitate production of a fusion protein comprising a PBRT or a PBRC.
  • such vectors can comprise one or more cloning sites between the sequence coding for the PBRT or the PBRC to enable generation of an in-frame translation product.
  • Such vectors may comprise multiple cloning sites in any of three reading frames. Methods for generating such expression vectors are well known in the art.
  • a variety of expression systems can be used to produce the PBRCs and PBRC linked purification moieties described herein.
  • Such expression systems include vector based expression systems.
  • Exemplary vector base expression systems suitable for use with the methods described herein include, but are not limited to, chromosomal, episomal and virus- derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from insertion elements, from yeast episomes, from viruses such as baculoviruses, retroviruses and vectors derived from combinations thereof such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • the expression system vectors may contain regulatory regions that regulate as well as engender expression.
  • any system or vector suitable to maintain, propagate or express polynucleotide or polypeptide in a host cell may be used for expression in this regard.
  • Expression systems and expression vectors can contain regulatory sequences that direct high level expression of foreign proteins relative to the growth of the host cell. Regulatory sequences are well known to those skilled in the art and examples include, but are not limited to, those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of regulatory elements in the vector, for example, enhancer sequences. Any of these could be used to construct chimeric genes for production of the any of the binding peptides of the present invention. These chimeric genes could then be introduced into appropriate microorganisms via transformation to provide high level expression of the peptides.
  • a number of recombinant expression vectors can be used for expression of the PBRCs and PBRC linked purification moieties described herein.
  • the PBRT linked purification moieties described herein can be expressed in bacterial cells such as E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells, amphibian cells, or mammalian cells. Suitable host cells are well known to one skilled in the art.
  • the recombinant expression vector can be transcribed and translated in vitro, using, for example T7 promoter regulatory sequences and T7 polymerase.
  • E. coli expression vectors include pTrc (Amann E et al., Gene 69:301-15 (1988)) and pET l id (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) pp. 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET 1 Id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman S, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) pp. 119-28).
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada K et al., Nucleic Acids Res. 20(Suppl.):2111-18 (1992)). Such alteration of nucleic acid sequences can be carried out by standard DNA synthesis techniques.
  • a nucleic acid can be expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed B, Nature 329:840-41 (1987)) and pMT2PC (Kaufman R J et al, EMBO J. 6: 187-95 (1987)).
  • the expression vector's control functions can be provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40.
  • a number of these methodologies can also be applied in vivo, systemically or locally, in a complex biological system such as a human.
  • a complex biological system such as a human.
  • increased copy number of nucleic acids PBRC or PBRC linked purification moieties described herein in expressible from (by DNA transfection) can be employed.
  • Nucleic acid purification moieties encoding PBRT or PBRC linked purification moieties described herein can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see for example, Gonzalez et al., Bioconjugate Chem. 10: 1068- 1074, 1999; Wang et al, International PCT Publication Nos. WO 03/47518 and WO
  • Purification moieties that can be linked to the PBRTs or PBRCs described herein can be any purification moiety, including a biologically active protein (e.g., a therapeutic peptide, protein or an enzyme useful in industrial biocatalysis).
  • a biologically active protein e.g., a therapeutic peptide, protein or an enzyme useful in industrial biocatalysis.
  • the purification moieties suitable for use with the methods described herein can be of widely varying types, including, for example, peptides, non-peptide proteins, lipids, oligonucleotides and carbohydrates, or alternatively a ligand-binding protein or an active fragment thereof having binding affinity to a molecule selected from the group consisting of small organic or inorganic molecules, proteins, peptides, single-stranded or double-stranded oligonucleotides, polynucleotides, lipids, and carbohydrates.
  • Suitable purification moieties include, but are not limited to, molecules useful in medicine, agriculture and other scientific and industrial fields.
  • suitable molecules include those of interest in medicine, agriculture or other scientific or industrial fields.
  • suitable proteins include enzymes utilized in replacement therapy;
  • polypeptides can be produced using the methods described herein.
  • the present invention is not limited to any specific types of recombinant polypeptide described herein. Instead, it encompasses any and all recombinant polypeptides.
  • the PBRTs or PBRCs described herein can be joined to a purification moiety from any source or origin and can include a polypeptide found in prokaryotes, viruses, and eukaryotes, including fungi, plants, yeasts, insects, and animals, including mammals (e.g. humans).
  • Purification moieties suitable for use with the methods described herein include, but are not limited to any polypeptide sequences, known or hypothetical or unknown, which can be identified using common sequence repositories. Examples of such sequence repositories, include, but are not limited to GenBank EMBL, DDBJ and the NCBI. Other repositories can easily be identified by searching on the internet.
  • Polypeptides that can be produced using the methods described herein also include polypeptides have at least about 60%, 70%, 75%, 80%, 90%, 95%, or at least about 99% or more identity to any known or available
  • polypeptide e.g., a therapeutic polypeptide, a diagnostic polypeptide, an industrial enzyme, or portion thereof, and the like.
  • Purification moieties suitable for use with the methods described herein include, but are not limited to, polypeptides comprising one or more non-natural amino acids.
  • Purification moieties suitable for use with the methods described herein include, but are not limited to, cytokines, inflammatory molecules, growth factors, their receptors, and oncogene products or portions thereof.
  • cytokines, inflammatory molecules, growth factors, their receptors, and oncogene products include, but are not limited to e.g., alpha- 1 antitrypsin, Angiostatin, Antihemo lytic factor, antibodies (including an antibody or a functional fragment or derivative thereof selected from: Fab, Fab', F(ab)2, Fd, Fv, ScFv, diabody, tribody, tetrabody, dimer, trimer or minibody), angiogenic molecules, angiostatic molecules, Apolipopolypeptide, Apopolypeptide, Asparaginase, Adenosine deaminase, Atrial natriuretic factor, Atrial natriuretic polypeptide, Atrial peptides, Angiotensin family members, Bone
  • polypeptide-1 alpha Monocyte inflammatory polypeptide- 1 beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262), CD40 ligand, C-kit Ligand, Ciliary
  • Neurotrophic Factor Collagen, Colony stimulating factor (CSF), Complement factor 5a, Complement inhibitor, Complement receptor 1, cytokines, (e.g., epithelial Neutrophil Activating Peptide-78, GRO alpha/MGSA, GRO beta , GRO gamma , MIP-1 alpha , MIP-1 delta, MCP-1), deoxyribonucleic acids, Epidermal Growth Factor (EGF), Erythropoietin ("EPO", representing a preferred target for modification by the incorporation of one or more non-natural amino acid), Exfoliating toxins A and B, Factor IX, Factor VII, Factor VIII, Factor X, Fibroblast Growth Factor (FGF), Fibrinogen, Fibronectin, G-CSF, GM-CSF, Glucocerebrosidase, Gonadotropin, growth factors, Hedgehog polypeptides (e.g., Sonic, Indian, Desert), Hemoglobin, Hepatocyte Growth
  • Keratinocyte Growth Factor Keratinocyte Growth Factor (KGF), Lactoferrin, leukemia inhibitory factor, Luciferase, Neurturin, Neutrophil inhibitory factor (NIF), oncostatin M, Osteogenic polypeptide, Parathyroid hormone, PD-ECSF, PDGF, peptide hormones (e.g., Human Growth Hormone), Oncogene products (Mos, Rel, Ras, Raf, Met, etc.), Pleiotropin, Polypeptide A, Polypeptide G, Pyrogenic exotoxins A, B, and C, Relaxin, Renin, ribonucleic acids, SCF/c-kit, Signal transcriptional activators and suppressors (p53, Tat, Fos, Myc, Jun, Myb, etc.), Soluble complement receptor 1, Soluble I-CAM 1, Soluble interleukin receptors (IL-1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15), soluble adhesion molecules, Soluble TNF receptor, Somato
  • Additional purification moieties suitable for use with the methods described herein include, but are not limited to, enzymes (e.g., industrial enzymes) or portions thereof.
  • enzymes include, but are not limited to amidases, amino acid racemases, acylases, dehalogenases, dioxygenases, diarylpropane peroxidases, epimerases, epoxide hydrolases, esterases, isomerases, kinases, glucose isomerases, glycosidases, glycosyl transferases, haloperoxidases, monooxygenases (e.g., p450s), lipases, lignin peroxidases, nitrile hydratases, nitrilases, proteases, phosphatases, subtilisins, transaminase, and nucleases.
  • amidases amino acid racemases, acylases, dehalogenases, dioxygenases, diarylpropane peroxidases, epimerases, epoxide hydrolases, esterases, isomerases, kinases, glucose isomerases, glycosida
  • such enzymes comprising a PBRT or PBRC can be used as immobilized enzymes in industrial biocatalysis.
  • the enzymes comprising a PBRTs or a PBRC can also be added to a solution to facilitate biocatalysis and then reisolated from the solution.
  • Additional purification moieties suitable for use with the methods described herein include, but are not limited to, agriculturally related polypeptides such as insect resistance polypeptides (e.g., Cry polypeptides), starch and lipid production enzymes, plant and insect toxins, toxin-resistance polypeptides, Mycotoxin detoxification polypeptides, plant growth enzymes (e.g., Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase), lipoxygenase, and Phosphoenolpyruvate carboxylase.
  • agriculturally related polypeptides such as insect resistance polypeptides (e.g., Cry polypeptides), starch and lipid production enzymes, plant and insect toxins, toxin-resistance polypeptides, Mycotoxin detoxification polypeptides, plant growth enzymes (e.g., Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase), lipoxygenase, and P
  • Additional purification moieties suitable for use with the methods described herein include, but are not limited to, antibodies, immunoglobulin domains of antibodies and their fragments.
  • antibodies include, but are not limited to antibodies, antibody fragments, antibody derivatives, Fab fragments, Fab' fragments, F(ab)2 fragments, Fd fragments, Fv fragments, single-chain Fv fragments (scFv), diabodies, tribodies, tetrabodies, dimers, trimers, and minibodies.
  • Additional purification moieties suitable for use with the methods described herein include, but are not limited to, prophylactic vaccine or therapeutic vaccine polypeptides.
  • a prophylactic vaccine is one administered to subjects who are not infected with a condition against which the vaccine is designed to protect.
  • a preventive vaccine will prevent a virus from establishing an infection in a vaccinated subject.
  • a prophylactic vaccine may still confer some protection to a subject.
  • a prophylactic vaccine may decrease the symptoms, severity, and/or duration of the disease.
  • a therapeutic vaccine is administered to reduce the impact of a viral infection in subjects already infected with that virus.
  • Vaccine polypeptides include polypeptides, or polypeptide fragments from infectious fungi (e.g., Aspergillus, Candida species) bacteria (e.g. E. coli, Staphylococci aureus)), or
  • Streptococci e.g., pneumoniae
  • protozoa such as sporozoa (e.g., Plasmodia), rhizopods (e.g., Entamoeba) and flagellates (Trypanosoma, Leishmania, Trichomonas, Giardia, etc.); viruses such as (+) RNA viruses
  • examples include Poxviruses e.g., vaccinia; Picornaviruses, e.g., polio; Togaviruses, e.g., rubella; Flaviviruses, e.g., HCV; and Coronaviruses), (-) RNA viruses (e.g., Rhabdoviruses, e.g., VSV; Paramyxovimses, e.g., RSV; Orthomyxovimses, e.g., influenza; Bunyaviruses; and Arenaviruses), dsDNA viruses (Reoviruses, for example
  • Additional purification moieties suitable for use with the methods described herein include, but are not limited to, molecules that comprise a chemical moiety selected from the group consisting of: cytotoxins, pharmaceutical drugs, dyes or fluorescent labels, a nucleophilic or electrophilic group, a ketone or aldehyde, azide or alkyne compounds, photocaged groups, tags, a peptide, a polypeptide, a polypeptide, an oligosaccharide, polyethylene glycol with any molecular weight and in any geometry, polyvinyl alcohol, metals, metal complexes, polyamines, imidizoles, carbohydrates, lipids, biopolymers, particles, solid supports, a polymer, a targeting agent, an affinity group, any agent to which a complementary reactive chemical group can be attached, biophysical or biochemical probes, isotypically-labeled probes, spin-label amino acids, fluorophores, aryl iodides and bromides.
  • cytotoxins cytotoxins
  • a variant PBRT is a PBRT
  • an amino acid having an evolutionary positive relatedness or an amino acid having an evolutionary neutral relatedness.
  • variant precipatable-beta roll cassette refers to an amino acid sequence comprising at least one variant PBRT.
  • a variant PBRC will comprise at least two variant PBRTs.
  • a variant PBRC will comprise at least 3 variant PBRTs, at least 4 variant PBRTs, at least 5 variant PBRTs, at least 6 variant PBRTs, at least 7 variant PBRTs, at least 8 variant PBRTs, at least 9 variant PBRTs, at least 10 variant PBRTs, at least 11 variant PBRTs, at least 12 variant PBRTs, at least 13 variant PBRTs, at least 14 variant PBRTs, at least 15 variant PBRTs, at least 16 variant PBRTs, at least 17 variant PBRTs, at least 18 variant PBRTs, at least 19 variant PBRTs, at least 20 variant PBRTs, or 20 or more variant PBRTs.
  • the PBRCs described herein will comprise a plurality of variant precipatable beta roll tags arranged in a tandem repeat.
  • the variant PBRCs described herein can comprise at least 2 variant PBRTs, at least 3 variant PBRTs, at least 4 variant PBRTs, at least 5 variant PBRTs, at least 6 variant PBRTs, at least 7 variant PBRTs, at least 8 variant PBRTs, at least 9 variant PBRTs, at least 10 variant PBRTs, at least 11 variant PBRTs, at least 12 variant PBRTs, at least 13 variant PBRTs, at least 14 variant PBRTs, at least 15 variant PBRTs, at least 16 variant PBRTs, at least 17 variant PBRTs, at least 18 variant PBRTs, at least 19 variant PBRTs, at least 20 variant PBRTs, or 20 or more variant PBRTs in tandem repeat.
  • a PBRC can comprise at least two PBRCs separated by a linking amino acid sequence.
  • a linking amino acid sequence in present between two PBRTs a PBRTs located at either end of the linking sequence can be an individual PBRT or it can be a PBRTs that is part of a tandem arrangement.
  • a variant PBRC will comprise at least 2 variant PBRCs, at least variant 3 PBRTs, at least 4 variant PBRTs, at least 5 variant PBRTs, at least 6 variant PBRTs, at least 7 variant PBRTs, at least 8 variant PBRTs, at least 9 variant PBRTs, at least 10 variant PBRTs, at least 11 variant PBRTs, at least 12 variant PBRTs, at least 13 variant PBRTs, at least 14 variant PBRTs, at least 15 variant PBRTs, at least 16 variant PBRTs, at least 17 variant PBRTs, at least 18 variant PBRTs, at least 19 variant PBRTs, at least 20 variant PBRTs, or 20 or more variant PBRTs, all of which are located N-terminally to a CS.
  • a variant PBRC will comprise at least 2 variant PBRCs, at least variant 3 PBRTs, at least 4 variant PBRTs, at least 5 variant PBRTs, at least 6 variant PBRTs, at least 7 variant PBRTs, at least 8 variant PBRTs, at least 9 variant PBRTs, at least 10 variant PBRTs, at least 11 variant PBRTs, at least 12 variant PBRTs, at least 13 variant PBRTs, at least 14 variant PBRTs, at least 15 variant PBRTs, at least 16 variant PBRTs, at least 17 variant PBRTs, at least 18 variant PBRTs, at least 19 variant PBRTs, at least 20 variant PBRTs, or 20 or more variant PBRTs, all of which are located C-terminally to a CS.
  • the invention relates to a variant PBRT that contains one or more amino acid insertions, deletions or substitutions as compared to the sequence of SEQ ID NO: 1 and wherein the variant PBRT retains an ability to bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the PBRT.
  • Changes can be introduced by mutation into nucleic acid sequences, thereby leading to changes in the amino acid sequence of the encoded protein, without altering the functional activity of a PBRT or a PBRC.
  • nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues can be made in the sequence of a PBRT or a PBRC.
  • a non-essential amino acid residue is a residue that can be altered from the sequence of an amino acid of this invention without altering the ability of the PBRT or PBRC to bind to bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the PBRT or PBRC.
  • Exemplary residues which are non-essential and therefore amenable to substitution to generate the variant PBRTs and PBRCs described herein can be identified by one of ordinary skill in the art by performing an amino acid alignment of two more PBRTs or PBRCs and determining residues that are not required for the PBRT or PBRC to bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the PBRT or PBRC.
  • Mutations can be introduced randomly along all or part of a nucleic acid sequence encoding a PBRT or PBRC, such as by saturation mutagenesis, and the resultant mutants can be screened, for example, for their ability bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT or variant PBRC.
  • the purification moiety linked to the variant PBRT or variant PBRC can be expressed recombinantly in a host cell and the functional activity of the precipatable beta-roll tag can be determined using assays available in the art for assessing binding to Ca2+, undergoing reversible precipitation in the presence of Ca2+, or inducing reversible precipitation of purification moiety linked to the variant PBRT or variant PBRC.
  • the variant PBRTs described herein can comprise one or more amino acid substitutions, insertions or deletions, wherein the variant PBRT is functionally equivalent a PBRT having the sequence GGAGNDTLY (SEQ ID No. 1).
  • the variant PBRT has an identical ability bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety in a manner similar to, but not necessarily identical to a PBRC comprising only PBRTs of the sequence GGAGNDTLY. In one embodiment, the variant PBRT has a reduced ability bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety in a manner similar to, but not necessarily identical to a PBRC comprising only PBRTs of the sequence GGAGNDTLY.
  • the variant PBRT has an increased ability bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety in a manner similar to, but not necessarily identical to a PBRC comprising only PBRTs of the sequence GGAGNDTLY.
  • the variant PBRCs described herein can also comprise on or more PBRTs in addition to one or more variant PBRTs.
  • the variant PBRCs described herein can also be employed in any embodiments or configuration described herein for a PBRC.
  • the description of a composition comprising a PBRC, or a method comprising a PBRC applies equally to a variant PBRT or a variant PBRC so long as the variant PBRT or the variant PBRC can bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety in a manner similar to, but not necessarily identical to a PBRC comprising only PBRTs of the sequence GGAGNDTLY.
  • a variant PBRT comprises the sequence GGXGXDXXX (SEQ ID NO: 2) wherein X can be selected from the group consisting of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.
  • the variant PBRT has a sequence of GGXGXDXXX (SEQ ID NO: 2), wherein X is not proline.
  • the variant PBRT comprises the sequence GGXGXDXXX (SEQ ID NO: 2) wherein X is a natural or non-natural amino acid comprising a modification.
  • a variant PBRT or PBRC comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, 99% identity with an amino acid sequence of SEQ ID NO: 1.
  • sequence identity means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions.
  • sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. Techniques for determining sequence identity are well known to one skilled in the art, and include, for example, analysis with a sequence comparison algorithm or FASTA version 3.0t78 using default parameters (Pearson and Lipman, Proc Natl Acad Sci U S A. 1988 Apr; 85(8):2444-8).
  • scoring of amino acid can be calculated using the PAM250 matrix as described in Dayhoff et al., (1978) in Atlas of Protein Sequence and Structure, ed. Dayhoff, M. (Natl. Biomed. Res. Found., Silver Spring, MD), Vol. 5, Suppl. 3, pp. 345-352.
  • Percent identity or percent similarity of a DNA or peptide sequence can be determined, for example, by comparing sequence information using the GAP computer program.
  • the GAP program utilizes the alignment method of Needleman et al., 1970, as revised by Smith et al., 1981. Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences.
  • the preferred parameters for the GAP program are the default parameters, which do not impose a penalty for end gaps. See e.g., Schwartz et al., 1979; Gribskov et al., 1986. Nucleic acids that differ due to degeneracy of the genetic code, and still encode the PBRTs or PBRCs, described herein are encompassed by the present disclosure.
  • Variants can be produced by any number of methods, including but not limited to, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, and any combination thereof.
  • Variant PBRTs or variant PBRCs falling within the scope of this invention can, in general, be generated by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the purification moiety at the target site, or (c) the bulk of the side chain.
  • a variant PBRT or a variant PBRC can comprise a conservative amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain configuration.
  • Amino acid residues having similar side chain configurations have been defined in the art within in accordance with the following categories: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), aromatic side chains (e.g., tyrosine,
  • a variant PBRT or a variant PBRC can comprise a conservative amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain group.
  • Amino acid residues having similar side chain groups have been defined in the art within in accordance with the following categories: a no side chain group (glycine), an aliphatic side chain group (alanine, valine, leucine, isoleucine, proline), a hydroxyl side chain group (serine, threonine), an acidic side chain group (aspartic acid, glutamic acid), an amide side chain group (asparagine, glutamine), a basic side chain group (lysine, arginine), an imidazole side chain group (histidine), an aromatic side chain group (phenylalanine, tyrosine, tryptophan), and a sulfur containing side chain group
  • a variant PBRT or a variant PBRC can comprise a conservative amino acid substitution in which an amino acid residue is replaced an amino acid having evolutionarily positive relatedness.
  • Amino acids having evolutionarily positive relatedness have been defined in the art as follows (wherein the amino acid(s) having evolutionarily positive relatedness are indicated in parentheses): Alanine (serine, threonine, proline, glycine); Arginine (glutamine, histidine, lysine, tryptophan); Asparagine (serine, threonine, aspartic acid, glutamic acid, glutamine, histidine, lysine); Aspartic acid (threonine, glycine, asparagine, glutamine, glutamic acid, histidine); Glutamic acid (threonine, asparagine, aspartic acid, glutamine, histidine); Glutamine (asparagine, aspartic acid, glutamic acid, histidine
  • Phenylalanine (isoleucine, leucine, tyrosine); Proline (serine, threonine, alanine); Serine (threonine, proline, alanine, glycine, asparagine); Threonine (serine, proline, alanine, glycine, asparagine, aspartic acid, glutamic acid, lysine, isoleucine, valine); Tryptophan (arginine, tyrosine); Tyrosine (phenylalanine, tryptophan); Valine (threonine, methionine, isoleucine, leucine) (see Dayhoff et al., (1978) in Atlas of Protein Sequence and Structure, ed. Dayhoff, M., Natl. Biomed. Res. Found., Silver Spring, MD), Vol. 5, Suppl. 3, pp. 345-352).
  • variant of a variant PBRT or a variant PBRC can comprise a conservative amino acid substitution in which an amino acid residue is replaced an amino acid having evolutionarily positive relatedness.
  • Amino acids having evolutionarily neutral relatedness have been defined in the art as follows (wherein the amino acid(s) having evolutionarily neutral relatedness are indicated in parentheses): Alanine (asparagine, aspartic acid, glutamine, glutamic acid, valine); Arginine (serine, proline, asparagine, methionine); Asparagine (alanine, glycine, arginine); Aspartic acid (serine, alanine, lysine); Cysteine (serine, tyrosine); Glutamic acid (serine, alanine, glycine, lysine); Glutamine (proline, alanine); Glycine (asparagine, glutamic acid); Histidine (proline, lysine),
  • the glycine at position 1 of SEQ ID NO: 1 is not mutated.
  • the glycine at position 1 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the glycine is replaced with an amino acid having an uncharged polar side chain configuration (e.g., asparagine, glutamine, serine, threonine, tyrosine, or cysteine).
  • an uncharged polar side chain configuration e.g., asparagine, glutamine, serine, threonine, tyrosine, or cysteine.
  • the glycine at position 1 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the glycine is replaced with an amino acid having an aliphatic side chain configuration (e.g., alanine, valine, leucine, isoleucine)
  • an amino acid having an aliphatic side chain configuration e.g., alanine, valine, leucine, isoleucine
  • the glycine at position 1 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily positive relatedness such that the glycine is replaced with any of serine, threonine, alanine, or aspartic acid.
  • the glycine at position 1 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily neutral relatedness such that the glycine is replaced with any of asparagine or glutamic acid.
  • mutation of the glycine at position 1 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid wherein mutation of the glycine at position 1 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
  • the glycine at position 2 of SEQ ID NO: 1 can be mutated to an asparagine residue.
  • the glycine at position 2 of SEQ ID NO: 1 can be mutated to an aspartic acid residue.
  • the glycine at position 2 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the glycine is replaced with an amino acid having an uncharged polar side chain configuration (e.g., asparagine, glutamine, serine, threonine, tyrosine, or cysteine).
  • an uncharged polar side chain configuration e.g., asparagine, glutamine, serine, threonine, tyrosine, or cysteine.
  • the glycine at position 2 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the glycine is replaced with an amino acid having an aliphatic side chain configuration (e.g., alanine, valine, leucine, isoleucine)
  • an amino acid having an aliphatic side chain configuration e.g., alanine, valine, leucine, isoleucine
  • the glycine at position 2 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily positive relatedness such that the glycine is replaced with any of serine, threonine, alanine, or aspartic acid.
  • the glycine at position 2 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily neutral relatedness such that the glycine is replaced with any of asparagine or glutamic acid.
  • mutation of the glycine at position 2 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid wherein mutation of the glycine at position 2 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
  • the alanine at position 3 of SEQ ID NO: 1 can be mutated to a serine, glycine, or aspartic acid residue.
  • the alanine at position 3 of SEQ ID NO: 1 can be mutated to a glutamic acid, leucine, or asparagine residue.
  • the alanine at position 3 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the alanine is replaced with an amino acid having a nonpolar side chain configuration (e.g., valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan).
  • an amino acid having a nonpolar side chain configuration e.g., valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan.
  • the alanine at position 3 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the alanine is replaced with an amino acid having an aliphatic side chain configuration (e.g., glycine, valine, leucine, isoleucine)
  • an amino acid having an aliphatic side chain configuration e.g., glycine, valine, leucine, isoleucine
  • the alanine at position 3 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain group such that the alanine is replaced with an amino acid having an aliphatic side chain group (e.g., valine, leucine, isoleucine, proline).
  • an amino acid having an aliphatic side chain group e.g., valine, leucine, isoleucine, proline.
  • the alanine at position 3 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily positive relatedness such that the alanine is replaced with any of serine, threonine, proline, or glycine.
  • the at position 3 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily neutral relatedness such that the alanine is replaced with any of asparagine, aspartic acid, glutamine, glutamic acid, or valine.
  • mutation of the alanine at position 3 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid wherein mutation of the alanine at position 3 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
  • the glycine at position 4 of SEQ ID NO: 1 can be mutated to an alanine residue.
  • the glycine at position 4 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the glycine is replaced with an amino acid having an uncharged polar side chain configuration (e.g., asparagine, glutamine, serine, threonine, tyrosine, or cysteine).
  • an uncharged polar side chain configuration e.g., asparagine, glutamine, serine, threonine, tyrosine, or cysteine.
  • the glycine at position 4 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the glycine is replaced with an amino acid having an aliphatic side chain configuration (e.g., alanine, valine, leucine, isoleucine)
  • an amino acid having an aliphatic side chain configuration e.g., alanine, valine, leucine, isoleucine
  • the glycine at position 4 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily positive relatedness such that the glycine is replaced with any of serine, threonine, alanine, or aspartic acid.
  • the glycine at position 4 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily neutral relatedness such that the glycine is replaced with any of asparagine or glutamic acid.
  • mutation of the glycine at position 4 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid wherein mutation of the glycine at position 4 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
  • the asparagine at position 5 of SEQ ID NO: 1 can be mutated to an aspartic acid or alanine residue.
  • the asparagine at position 5 of SEQ ID NO: 1 can be mutated to a serine residue.
  • the asparagine at position 5 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the asparagine is replaced with an amino acid having an uncharged polar side chain configuration (e.g., glycine, glutamine, serine, threonine, tyrosine, cysteine)
  • an amino acid having an uncharged polar side chain configuration e.g., glycine, glutamine, serine, threonine, tyrosine, cysteine
  • the asparagine at position 5 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the asparagine is replaced with an amino acid having a the side chain configuration of its amide (e.g., aspartic acid).
  • the asparagine at position 5 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain group such that the asparagine is replaced with an amino acid having an amide side chain group (e.g., glutamine).
  • an amide side chain group e.g., glutamine
  • the asparagine at position 5 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily positive relatedness such that the asparagine is replaced with any of serine, threonine, aspartic acid, glutamic acid, glutamine, histidine, or lysine.
  • the asparagine at position 5 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily neutral relatedness such that the asparagine is replaced with any of alanine, glycine, or arginine.
  • mutation of the asparagine at position 5 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid wherein mutation of the asparagine at position 5 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
  • the aspartic acid at position 6 of SEQ ID NO: 1 can be mutated to an asparagine residue.
  • the aspartic acid at position 6 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the aspartic acid is replaced with an amino acid having an acidic side chain configuration (e.g., glutamic acid).
  • an amino acid having an acidic side chain configuration e.g., glutamic acid
  • the aspartic acid at position 6 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the aspartic acid is replaced with an amino acid having a the side chain configuration of its amide (e.g., asparagine).
  • an amino acid having a the side chain configuration of its amide e.g., asparagine
  • the aspartic acid at position 6 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain group such that the aspartic acid is replaced with an amino acid having an acidic side chain group (e.g., glutamic acid).
  • an amino acid having an acidic side chain group e.g., glutamic acid
  • the aspartic acid at position 6 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily positive relatedness such that the aspartic acid is replaced with any of threonine, glycine, asparagine, glutamine, glutamic acid, or histidine.
  • the aspartic acid at position 6 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily neutral relatedness such that the aspartic acid is replaced with any of serine, alanine, or lysine.
  • mutation of the aspartic acid at position 6 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid wherein mutation of the aspartic acid at position 6 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
  • the threonine at position 7 of SEQ ID NO: 1 can be mutated to an isoleucine or valine residue.
  • the threonine at position 7 of SEQ ID NO: 1 can be mutated to a leucine residue.
  • the threonine at position 7 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the threonine is replaced with an amino acid having an uncharged polar side chain configuration (e.g., glycine, asparagine, glutamine, serine, tyrosine, or cysteine).
  • an uncharged polar side chain configuration e.g., glycine, asparagine, glutamine, serine, tyrosine, or cysteine.
  • the threonine at position 7 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the threonine is replaced with an amino acid having a beta-branched side chain configuration (e.g., valine, isoleucine).
  • an amino acid having a beta-branched side chain configuration e.g., valine, isoleucine
  • the threonine at position 7 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain group such that the threonine is replaced with an amino acid having an hydroxyl side chain group (e.g., serine).
  • an amino acid having an hydroxyl side chain group e.g., serine
  • the threonine at position 7 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily positive relatedness such that the threonine is replaced with any of serine, proline, alanine, glycine, asparagine, aspartic acid, glutamic acid, lysine, isoleucine, or valine [00185]
  • mutation of the threonine at position 7 of SEQ ID NO: 1 to any of glycine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, valine wherein mutation of the threonine at position 7 of SEQ ID NO: 1 will result in a precip
  • mutation of the threonine at position 7 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid wherein mutation of the threonine at position 7 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
  • the leucine at position 8 of SEQ ID NO: 1 can be mutated to an isoleucine residue.
  • the leucine at position 8 of SEQ ID NO: 1 can be mutated to a phenylalanine residue.
  • the leucine at position 8 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the leucine is replaced with an amino acid having a nonpolar side chain configuration (e.g., alanine, valine, isoleucine, proline, phenylalanine, methionine, or tryptophan).
  • an amino acid having a nonpolar side chain configuration e.g., alanine, valine, isoleucine, proline, phenylalanine, methionine, or tryptophan.
  • the leucine at position 8 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the leucine is replaced with an amino acid having an aliphatic side chain configuration (e.g., glycine, alanine, valine, or isoleucine).
  • an amino acid having an aliphatic side chain configuration e.g., glycine, alanine, valine, or isoleucine.
  • leucine at position 8 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain group such that the leucine is replaced with an amino acid having an aliphatic side chain group (e.g., alanine, valine, isoleucine, proline).
  • an amino acid having an aliphatic side chain group e.g., alanine, valine, isoleucine, proline.
  • the leucine at position 8 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily positive relatedness such that the leucine is replaced with any of methionine, isoleucine, valine, or phenylalanine [00193]
  • the leucine at position 8 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily neutral relatedness such that the leucine is replaced with any of serine, asparagine, glutamic acid, histidine, or methionine.
  • mutation of the leucine at position 8 of SEQ ID NO: 1 non-natural amino acid wherein mutation of leucine at position 8 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
  • the tyrosine at position 9 of SEQ ID NO: 1 can be mutated to an isoleucine or valine residue.
  • the tyrosine at position 9 of SEQ ID NO: 1 can be mutated to a phenylalanine, threonine, asparagine, aspartic acid, lysine, or serine residue.
  • the tyrosine at position 9 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the tyrosine is replaced with an amino acid having an uncharged polar side chain configuration (e.g., glycine, asparagine, glutamine, serine, threonine, or cysteine).
  • an uncharged polar side chain configuration e.g., glycine, asparagine, glutamine, serine, threonine, or cysteine.
  • the tyrosine at position 9 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the tyrosine is replaced with an amino acid having an aromatic side chain configuration (e.g., tyrosine, phenylalanine, tryptophan, or histidine).
  • an aromatic side chain configuration e.g., tyrosine, phenylalanine, tryptophan, or histidine.
  • the tyrosine at position 9 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain group such that the tyrosine is replaced with an amino acid having an aromatic side chain group (e.g., phenylalanine, tryptophan).
  • an aromatic side chain group e.g., phenylalanine, tryptophan
  • the tyrosine at position 9 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily positive relatedness such that the tyrosine is replaced with any of phenylalanine or tryptophan.
  • the tyrosine at position 9 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily neutral relatedness such that the tyrosine is replaced with any of cysteine or histidine.
  • mutation of the tyrosine at position 9 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid wherein mutation of the tyrosine at position 9 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
  • a variant PBRC can further comprise a capping sequence.
  • the capping sequence in a variant PBRC can be a variant capping sequence.
  • a variant capping sequence can be an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identity to SEQ ID NO: 3.
  • a variant capping sequence is an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identity to any of SEQ ID NO: 4-23.
  • a variant capping sequence is a sequence comprising one or more amino acid substitutions with an amino acid having a similar side chain group.
  • the variant capping sequence comprises the sequence of SEQ ID NO: 3 or any of SEQ ID NO: 4-23, wherein at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53,
  • a variant capping sequence is a sequence comprising one or more amino acid substitutions with an amino acid having a similar side chain configuration.
  • the variant capping sequence comprises the sequence of SEQ ID NO: 3 or any of SEQ ID NO: 6-23, wherein at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53,
  • a variant capping sequence is a sequence comprising one or more amino acid substitutions with an amino acid having evolutionarily positive relatedness.
  • the variant capping sequence comprises the sequence of SEQ ID NO: 3 or any of SEQ ID NO: 6-23, wherein at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53,
  • a variant capping sequence is a sequence comprising one or more amino acid substitutions with an amino acid having evolutionarily neutral relatedness.
  • the variant capping sequence comprises the sequence of SEQ ID NO: 3 or any of SEQ ID NO: 6-23, wherein at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53
  • the PBRT variants or PBRC variants described herein can also comprise a non-natural amino acid.
  • a non-natural amino acid can be, but is not limited to, an amino acid comprising a moiety where a chemical moiety is attached, such as an aldehyde- or keto-derivatized amino acid, or a non-natural amino acid that includes a chemical moiety.
  • a non-natural amino acid can also be an amino acid comprising a moiety where a saccharide moiety can be attached, or an amino acid that includes a saccharide moiety.
  • non-classical amino acids suitable for use with the methods and compositions described herein include, but are not limited to, D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, gamma-Abu, epsilon-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,
  • PBRT variants or PBRC variants described herein can also comprise one or more amino acid analog substitutions, e.g., unnatural amino acids such as alpha alpha- disubstituted amino acids, N-alkyl amino acids, lactic acid, and the like.
  • analogs include phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma- carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, 1,2,3,4,- tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citruline, . alpha.
  • PBRTs or PBRCs comprising an analog substitutions to bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the PBRT or PBRC using methods known to those skilled in the art.
  • PBRT variants or PBRC variants described herein can further comprise polypeptide analogs, such as peptide mimetics (Fauchere J, Adv. Drug Res. 15:29 (1986); Veber D F and Freidinger R M, Trends Neurosci. 8:392-96 (1985); Evans B E et al, J. Med. Chem 30: 1229-39 (1987)).
  • polypeptide analogs such as peptide mimetics
  • peptidomimetics are structurally similar to a template polypeptide (i.e., a polypeptide that has a biological or pharmacological activity), such as the PBRTs or PBRCs described herein, but have one or more peptide linkages replaced by a linkage selected from the group consisting of: ⁇ CH.sub.2NH ⁇ , ⁇ CH.sub.2S ⁇ ,—CH.sub.2— CH.sub.2-, ⁇ CH.dbd.CH ⁇ (cis and trans), -COCH.sub.2-, -CH(OH)CH.sub.2-, and - CH.sub.2SO— , by methods known in the art and further described in the following references: Spatola A F in "Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins," B.
  • Non-peptide linkage is—CH.sub.2NH-.
  • Such polypeptide mimetics can have advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.
  • Labeling of peptidomimetics can involve covalent attachment of one or more labels, directly or through a spacer (e.g., an amide group), to non-interfering position(s) on the peptidomimetic that are predicted by quantitative structure-activity data and/or molecular modeling.
  • Such non- interfering positions can be positions that do not from direct contacts with the
  • peptidomimetics can be done without substantially interfering with the desired biological or pharmacological activity of the peptidomimetic.
  • the ability of any peptidomimetics to polypeptides can be assayed for the ability to bind 1,4,- benzothiazepine or derivatives thereof using methods know to those skilled in the art.
  • Example 1 Purification of PBRT or PBRC linked Purification moieties
  • Polypeptides comprising a 5 or 17 repeat C-capped precipatable beta-roll tags were expressed as MBP fusion proteins. 50 mM calcium was added to clarified cell ly sates to induce precipitation of the polypeptides comprising a 5 or 17 repeat C-capped precipatable beta-roll tags. The precipitate was pelleted by centrifugation and the pellet was washed once and resuspended in buffer with 50 mM EGTA.
  • An intein domain can be coupled to the construct so that the cleavage reaction and subsequent second precipitation can be examined.
  • Other proteins, in addition to maltose binding protein can be used with the purification protocols described herein.
  • Example 2 PBRT sequence heat map.
  • the heat map for the precipatable beta-roll tag sequences described herein was determined by using BLAST to find beta roll sequences similar to the metalloprotease of S. marcescens and then quantifying the frequency of amino acids at each of the nine positions after beta roll sequences were identified ( Figure 4). Certain positions in the precipatable beta- roll tag are not highly variable (e.g. positions 1 , 2, 4, 6, and 8), whereas other positions, exhibit moderate conservation. Positions 7, 9 are highly variable and can be substituted with any natural or non-natural amino acid.
  • Example 3 Exemplary PBRCs

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Abstract

Ca2+ precipatable polypeptide tags and cassettes useful for purification of molecules from heterogeneous samples are disclosed. Methods for bioseparation of molecules comprising Ca2+ precipatable tags and cassettes are also disclosed.

Description

PRECIPATABLE PEPTIDES
[0001] This invention was made with government support under grant number W9132T-08- 2-0012 awarded by the DTRA and under grant number W9132T-08-2-0002 awarded by the US Army. The government has certain rights in the invention.
[0002] This application claims the benefit of and priority to U.S. provisional application Ser. No.: 61/475,042 filed April 13, 2011, and also claims the benefit of and priority to U.S. provisional application Ser. No.: 61/616,341 filed March 27, 2012, the disclosures of all of which are hereby incorporated by reference in their entireties for all purposes.
[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
[0004] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described herein.
BACKGROUND OF THE INVENTION
[0005] Rapid protein purification is an important requirement in many bioengineering applications where significant amounts of time are currently spent purifying proteins from heterogeneous samples. There are currently a number of approaches for performing bioseparation, but these approaches are expensive, time consuming, can require specialized treatments.
[0006] A variety of approaches currently exist for purifying recombinant proteins such as using a poly-histidine tag, glutathione S-transferase (GST) fusions or fusion to an elastin-like peptide (ELP). In the case of ELPs, a fusion protein can be precipitated from solution by increasing the temperature of the sample (Banki, et al., Nat Meth, vol. 2, no. 9, pp. 659-662, 2005; Fong et al, Trends in Biotechnology, vol. 28, no. 5, pp. 272-279, May. 2010). One limitation of ELP technology is that increased temperature can adversely affect the stability of fusion proteins. Another limitation of ELP technology is that inducing temperature changes are difficult in large scale preparations. [0007] There is a need for improved purification methods for rapid purification of molecules (e.g. exogenously expressed proteins) from heterogeneous samples in a rapid manner and with high levels of recovery. This invention addresses these needs.
SUMMARY OF THE INVENTION
[0008] In certain aspects, the invention relates to a precipatable beta roll cassette (PBRC) comprising one or more beta roll tags (PBRTs) wherein the one or more PBRTs comprise the amino acid sequence of SEQ ID NO: 1
[0009] In certain aspects, the invention relates to a precipatable beta roll cassette (PBRC) comprising one or more beta roll tags (PBRTs) wherein the one or more PBRTs are independently any of: (a) a polypeptide having the amino acid sequence of SEQ ID NO: 1, or (b) a polypeptide having the amino acid sequence of any of SEQ ID NOs: 25-1337
[0010] In certain aspects, the invention relates to a precipatable beta roll cassette (PBRC) comprising one or more beta roll tags (PBRTs) wherein the one or more PBRTs are independently any of: (a) a polypeptide having the amino acid sequence of SEQ ID NO: 1, or (b) a polypeptide having the amino acid sequence of any of SEQ ID NOs 25-1337, (c) a polypeptide comprising the amino acid sequence GXXXXXXXX, wherein, (i) the X at position 2 is an amino acid selected from the group consisting of glycine, asparagine or aspartic acid, and (ii) the X at position 3 is an amino acid selected from the group consisting of alanine, serine, glycine, aspartic acid, glutamic acid, leucine or asparagine, and (iii) the X at position 4 is an amino acid selected from the group consisting of glycine or alanine, and (iv) the X at position 5 is an amino acid selected from the group consisting of asparagine, aspartic acid, alanine, or serine, and (v) the X at position 6 is an amino acid selected from the group consisting of aspartic acid or asparagine, (vi) the X at position 7 is an amino acid selected from the group consisting of threonine, isoleucine, valine, or leucine, and (vii) the X at position 8 is an amino acid selected from the group consisting of leucine, isoleucine, or phenylalanine, and (viii) the X at position 9 is an amino acid selected from the group consisting of tyrosine, isoleucine, valine, phenylalanine, threonine, asparagine, aspartic acid, lysine or serine.
[0011] In certain aspects, the invention relates to a precipatable beta roll cassette (PBRC) comprising one or more beta roll tags (PBRTs) wherein the one or more PBRTs are independently any of: (a) a polypeptide having the amino acid sequence of SEQ ID NO: 1, or (b) a polypeptide having the amino acid sequence of any of SEQ ID NOs 25-1337, (c) a polypeptide comprising the amino acid sequence GXXXXXXXX, wherein, (i) the X at position 2 is an amino acid selected from the group consisting of glycine, asparagine or aspartic acid, and (ii) the X at position 3 is an amino acid selected from the group consisting of alanine, serine, glycine, aspartic acid, glutamic acid, leucine or asparagine, and (iii) the X at position 4 is an amino acid selected from the group consisting of glycine or alanine, and (iv) the X at position 5 is an amino acid selected from the group consisting of asparagine, aspartic acid, alanine, or serine, and (v) the X at position 6 is an amino acid selected from the group consisting of aspartic acid or asparagine, (vi) the X at position 7 is an amino acid selected from the group consisting of threonine, isoleucine, valine, or leucine, and (vii) the X at position 8 is an amino acid selected from the group consisting of leucine, isoleucine, or phenylalanine, and (viii) the X at position 9 is an amino acid selected from the group consisting of tyrosine, isoleucine, valine, phenylalanine, threonine, asparagine, aspartic acid, lysine or serine, or (c) a variant PBRT.
[0012] In certain aspects, the invention relates to a precipatable beta roll cassette (PBRC) comprising one or more beta roll tags (PBRTs) wherein the one or more PBRTs are independently any of: (a) a polypeptide having the amino acid sequence of SEQ ID NO: 1, or (b) a polypeptide having the amino acid sequence of any of SEQ ID NOs 25-1337, (c) a polypeptide comprising the amino acid sequence GXXXXXXXX, wherein, (i) the X at position 2 is an amino acid selected from the group consisting of glycine, asparagine or aspartic acid, and (ii) the X at position 3 is an amino acid selected from the group consisting of alanine, serine, glycine, aspartic acid, glutamic acid, leucine or asparagine, and (iii) the X at position 4 is an amino acid selected from the group consisting of glycine or alanine, and (iv) the X at position 5 is an amino acid selected from the group consisting of asparagine, aspartic acid, alanine, or serine, and (v) the X at position 6 is an amino acid selected from the group consisting of aspartic acid or asparagine, (vi) the X at position 7 is an amino acid selected from the group consisting of threonine, isoleucine, valine, or leucine, and (vii) the X at position 8 is an amino acid selected from the group consisting of leucine, isoleucine, or phenylalanine, and (viii) the X at position 9 is an amino acid selected from the group consisting of tyrosine, isoleucine, valine, phenylalanine, threonine, asparagine, aspartic acid, lysine or serine, or (c) a variant PBRT, wherein the PBRC further comprises a capping sequence.
[0013] In certain aspects, the invention relates to a precipatable beta roll cassette (PBRC) comprising one or more beta roll tags (PBRTs) wherein the one or more PBRTs are independently any of: (a) a polypeptide having the amino acid sequence of SEQ ID NO: 1, or (b) a polypeptide having the amino acid sequence of any of SEQ ID NOs 25-1337, (c) a polypeptide comprising the amino acid sequence GXXXXXXXX, wherein, (i) the X at position 2 is an amino acid selected from the group consisting of glycine, asparagine or aspartic acid, and (ii) the X at position 3 is an amino acid selected from the group consisting of alanine, serine, glycine, aspartic acid, glutamic acid, leucine or asparagine, and (iii) the X at position 4 is an amino acid selected from the group consisting of glycine or alanine, and (iv) the X at position 5 is an amino acid selected from the group consisting of asparagine, aspartic acid, alanine, or serine, and (v) the X at position 6 is an amino acid selected from the group consisting of aspartic acid or asparagine, (vi) the X at position 7 is an amino acid selected from the group consisting of threonine, isoleucine, valine, or leucine, and (vii) the X at position 8 is an amino acid selected from the group consisting of leucine, isoleucine, or phenylalanine, and (viii) the X at position 9 is an amino acid selected from the group consisting of tyrosine, isoleucine, valine, phenylalanine, threonine, asparagine, aspartic acid, lysine or serine, or (c) a variant PBRT, wherein the PBRC further comprises a stabilizing polypeptide.
[0014] In certain aspects, the invention relates to a PBRC linked purification moiety comprising a precipatable beta roll cassette (PBRC) comprising one or more beta roll tags (PBRTs) wherein the one or more PBRTs are independently any of: (a) a polypeptide having the amino acid sequence of SEQ ID NO: 1, or (b) a polypeptide having the amino acid sequence of any of SEQ ID NOs 25-1337, (c) a polypeptide comprising the amino acid sequence GXXXXXXXX, wherein, (i) the X at position 2 is an amino acid selected from the group consisting of glycine, asparagine or aspartic acid, and (ii) the X at position 3 is an amino acid selected from the group consisting of alanine, serine, glycine, aspartic acid, glutamic acid, leucine or asparagine, and (iii) the X at position 4 is an amino acid selected from the group consisting of glycine or alanine, and (iv) the X at position 5 is an amino acid selected from the group consisting of asparagine, aspartic acid, alanine, or serine, and (v) the X at position 6 is an amino acid selected from the group consisting of aspartic acid or asparagine, (vi) the X at position 7 is an amino acid selected from the group consisting of threonine, isoleucine, valine, or leucine, and (vii) the X at position 8 is an amino acid selected from the group consisting of leucine, isoleucine, or phenylalanine, and (viii) the X at position 9 is an amino acid selected from the group consisting of tyrosine, isoleucine, valine, phenylalanine, threonine, asparagine, aspartic acid, lysine or serine, or (c) a variant PBRT. In certain embodiments, the PBRC is linked to the purification moiety by a peptide bond. In certain embodiments, the PBRC is linked to the purification moiety by a chemical bond that is not a peptide bond.
[0015] In certain aspects, the invention relates to a PBRC linked purification moiety comprising a precipatable beta roll cassette (PBRC) comprising one or more beta roll tags (PBRTs) wherein the one or more PBRTs are independently any of: (a) a polypeptide having the amino acid sequence of SEQ ID NO: 1, or (b) a polypeptide having the amino acid sequence of any of SEQ ID NOs 25-1337, (c) a polypeptide comprising the amino acid sequence GXXXXXXXX, wherein, (i) the X at position 2 is an amino acid selected from the group consisting of glycine, asparagine or aspartic acid, and (ii) the X at position 3 is an amino acid selected from the group consisting of alanine, serine, glycine, aspartic acid, glutamic acid, leucine or asparagine, and (iii) the X at position 4 is an amino acid selected from the group consisting of glycine or alanine, and (iv) the X at position 5 is an amino acid selected from the group consisting of asparagine, aspartic acid, alanine, or serine, and (v) the X at position 6 is an amino acid selected from the group consisting of aspartic acid or asparagine, (vi) the X at position 7 is an amino acid selected from the group consisting of threonine, isoleucine, valine, or leucine, and (vii) the X at position 8 is an amino acid selected from the group consisting of leucine, isoleucine, or phenylalanine, and (viii) the X at position 9 is an amino acid selected from the group consisting of tyrosine, isoleucine, valine, phenylalanine, threonine, asparagine, aspartic acid, lysine or serine, or (c) a variant PBRT, wherein the PBRC further comprises a cleavage site located N-terminally or C-terminally to one or more of the one or more PBRTs. In certain embodiments, the cleavage site is selected from the group comprising an intein cleavage site, a Factor Xa cleavage site, a thrombin cleavage site, an enterokinase cleavage site, or a signal peptidase cleavage site.
[0016] In certain aspects, the invention relates to a polypeptide comprising a PBRC linked purification moiety comprising a precipatable beta roll cassette (PBRC) comprising one or more beta roll tags (PBRTs) wherein the one or more PBRTs are independently any of: (a) a polypeptide having the amino acid sequence of SEQ ID NO: 1, or (b) a polypeptide having the amino acid sequence of any of SEQ ID NOs 25-1337, (c) a polypeptide comprising the amino acid sequence GXXXXXXXX, wherein, (i) the X at position 2 is an amino acid selected from the group consisting of glycine, asparagine or aspartic acid, and (ii) the X at position 3 is an amino acid selected from the group consisting of alanine, serine, glycine, aspartic acid, glutamic acid, leucine or asparagine, and (iii) the X at position 4 is an amino acid selected from the group consisting of glycine or alanine, and (iv) the X at position 5 is an amino acid selected from the group consisting of asparagine, aspartic acid, alanine, or serine, and (v) the X at position 6 is an amino acid selected from the group consisting of aspartic acid or asparagine, (vi) the X at position 7 is an amino acid selected from the group consisting of threonine, isoleucine, valine, or leucine, and (vii) the X at position 8 is an amino acid selected from the group consisting of leucine, isoleucine, or phenylalanine, and (viii) the X at position 9 is an amino acid selected from the group consisting of tyrosine, isoleucine, valine, phenylalanine, threonine, asparagine, aspartic acid, lysine or serine, or (c) a variant PBRT; and a purification moiety.
[0017] In certain aspects, the invention relates to a nucleic acid encoding any of the polypeptides described herein.
[0018] In certain embodiments, the invention relates to a method for purifying a PBRC linked purification moiety, the method comprising (a) expressing the PBRC linked purification moiety in an expression system, (b) collecting the PBRC linked purification moiety in a first medium, (c) adding Ca2+ to the first medium so as to induce precipitation of PBRC linked purification moiety, (d) removing unprecipiated material from the medium from the precipitated PBRC linked purification moiety, (e) resuspending the PBRC linked purification moiety in a second medium having a lower than the free Ca2+ concentration than the free Ca2+ concentration obtained after step (c). In certain embodiments, a calcium chelator is added to the second medium of step (e). In certain embodiments, steps (c) to (e) are repeated one or more times. In certain embodiments, the method further comprises a step of removing precipitated material between step (b) and step (c). In certain embodiments, the PBRC comprises a cleavage site between the PBRC and the purification moiety. In certain embodiments, the method further comprises steps of: (i) cleaving the PBRC linked purification moiety so as to separate the purification moiety from the PBRC, (ii) adding Ca2+ to the medium so as to induce precipitation of the PBRC, and (iii) isolating the unprecipiated purification moiety. In certain embodiments, the cleavage site is an intein cleavage site.
[0019] In certain aspects, the invention relates to an expression vector comprising, as arranged from 5' to 3', a promoter, a nucleic acid sequence encoding the PBRC of any of claims 1-4, and at least one cloning site. [0020] In certain aspects, the invention relates to an expression vector comprising, as arranged from 5' to 3', a promoter, at least one cloning site, and a nucleic acid sequence encoding the PBRC of any of claims 1-4.
[0021] In certain aspects, the invention relates to an expression vector comprising, as arranged from 5' to 3', a promoter, at least one cloning site, a nucleic acid sequence encoding the PBRC of any of claims 1-4 and at least a second cloning site.
BRIEF DESCRIPTION OF THE FIGURES
[0022] Figure 1 shows a SDS-PAGE gel showing purification of a molecule comprising a precipatable beta-roll tag. Figure 1 A shows purification of precipitatable maltose binding protein comprising a precipitatable beta roll tag and an enterokinase cleavage site (MBP- PBRT). Total lysate is shown in lane 1 and lanes 2-7 are precipitation/ wash cycles. After two cycles, the sample consists nearly only of the MBP fusion protein with the precipitating tag attached (MBP-PBRT). Recovery is nearly 100% of the expressed protein. Figure IB shows SDS-PAGE analysis of purified MBP-PBRT and MBP-PBRT subjected to digestion of the enterokinase digestion site (Figure IB). Lane 1 shows purified MBP-PBRT, lane 2 shows supernatant after overnight digest and lane 3 shows the pellet.
[0023] Figure 2 shows a SDS-PAGE gel showing a successful purification of a polypeptide comprising a 5 or 17 repeat C-capped precipatable beta-roll tags. Lanes (from left to right): 1. MBP-5cap lysate, 2. MBP-5cap supernatant, 3. MBP-5C resuspended precipitate. The 4-6 lanes are the same expect with the capped 17 repeat construct.
[0024] Figure 3 is circular dichroism data (CD) showing precipitation of a polypeptide comprising a 17 repeat C-capped precipatable beta-roll tag
(GGAGNDTLY) 17INAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEII HAANQAVDQAGIEKLVEAMAQYPD (SEQ ID NO: 1344) out of solution with increasing calcium concentrations from 0 to 100 mM calcium. Loss of spectra indicates that the peptide is precipitating out of solution and no longer visible via CD.
[0025] Figure 4 shows a GGXGXDXXX (SEQ ID NO: 2) sequence heat map. The heat map was determined by using BLAST to find beta roll sequences similar to the metalloprotease of S. marcescens and then quantifying the frequency of amino acids at each of the nine positions after beta roll sequences were identified. See SEQ ID NO: 1343. [0026] Figure 5 shows a schematic illustration of the corkscrew configuration of tandem Ca2+ binding sequences. The figure shows a crystal structure image of the beta roll domain from the metalloprotease of S. marcescens (1SAT in the Protein Databank). Alternating 9- amino acid repeats are highlighted in green and red. Coordinated calcium ions are in white. The image represents 5 repeats of beta roll sequence. While there is no crystal structure for the adenylate cyclase beta roll domain, the high degree of sequence similarity to the consensus beta roll indicates that the adenylate cyclase beta roll domain is similar.
[0027] Figure 6 shows a schematic illustration of the corkscrew configuration of tandem Ca2+ binding sequences from a different angle than shown in Figure 5. The 6th residue binds the calcium ion. The 7th and 9th residues of each repeat are those that face outwards. The 8th residue is buried in the hydrophobic core. These residues are threonine and tyrosine, respectively in SEQ ID NO: 1.
[0028] Figure 7 shows the full crystal structure of the metalloprotease of S. marcescens. The black spheres indicate the position of the calcium ions within the beta roll domain.
[0029] Figure 8 shows a characterization of beta roll distribution, sequence deviation and number of repeats. Figure 8 A shows a distribution of beat roll lengths as frequency plotted against the number of beta roll repeats. Figure 8B shows beta roll sequence deviation from consensus by position plotted as proportion deviation as a function of distance from terminus. Figure 8C shows a probability of deviation from consensus plotted as probability as a function of the number of beta roll repeats. Figure 8D shows amount of beta roll in overall protein plotted as the number of beta roll residues as a function of the number of total residues.
[0030] Figure 9 shows an exemplary protocol for purification of a polypeptide comprising a precipatable beta roll tag. The images depict precipitation of maltose binding protein fused to seventeen repeats of a PBRT. The PBRC comprises 17 repeats of the amino acid sequence of SEQ ID NO: 1. The PBRC does not comprise a capping sequence.
[0031] Figure 10 shows purification of a maltose binding protein/PBRT/green fluorescent protein fusion (MBP-PBRT-GFP). Figure 10A shows a precipitation and resuspension of the MBP-PBRT-GFP polypeptide. Figure 10B shows SDS-PAGE of multiple precipitation wash cycles. The lanes of the SDS gel are as follows: lane 1 - ladder; lane 2 - clarified lysate; lane 3 - precipitation in 25 mM calcium followed by three washes; lane 4 - precipitation in 50 mM calcium followed by three washes; lane 5 - precipitation in 75 mM calcium followed by three washes; lane - 6 precipitation in 100 mM calcium followed by three washes. All lanes are normalized in terms of concentration. Recovery percentage can be estimated by comparing the band intensity in lane 2 to subsequent lanes.
[0032] Figure 11 shows non-limiting examples of polypeptides comprising a PBRT suitable for use with the methods described herein.
DETAILED DESCRIPTION OF THE INVENTION
[0033] The issued patents, applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.
[0034] Purification is a major requirement in many bioengineering applications where significant amounts of time are currently spent purifying proteins from heterogeneous samples. The invention described herein relates to methods for rapidly purifying a purification moiety (e.g. a target polypeptide) from a heterogeneous medium using a PBRC. For example, in certain embodiments, a target polypeptide can be produced as a fusion protein in frame with a PBRC. In certain embodiments, the fusion protein comprising the target protein and the PBRC can further comprise a specific cleavage site (e.g. an intein cleavage site or an enterokinase cleavage site) between the target protein sequence and the PBRC sequence. In such embodiments, cleavage at the cleavage site can be used to separate the PBRC from the target polypeptide.
[0035] The beta-roll domain is a right-handed beta helix found in a number of proteins. The consensus sequence for beta-roll peptides is tandem repeats of the 9 amino acid sequence GGXGXDX(L/F/I)X (SEQ ID NO: 24). In the presence of calcium, the conformation aligns to adopt the helical turns. Two repeats of the sequence are required to make a complete helical turn and each of these turns binds a calcium atom. In the absence of calcium, the peptide exists in a disordered conformation. Therefore the β-roll domain exhibits natural allosteric regulation. A synthetic version of the β-roll peptide has been produced with 8 repeats of GGSGNDNLS (SEQ ID NO: 1338) and this peptide was found to bind calcium and fold into the β-roll structure (Lilie et al, FEBS Lett 470 (2), 173 (2000)). The domain is capable of reversibly unfolding upon removal of the calcium. Beta roll sequences are known to play a role in secretion as part of the bacterial Type I secretion system (Davidson, et al., Microbiol. Mol. Biol. Rev. 72 (2008), pp. 317-364; Holland et al, Mol. Membr. Biol. 22 (2005), pp. 29-39; Chenal, et al, J. Biol. Chem. 284(2009), pp. 1781-1789; Welch, Pore- Forming Toxins 257 (2001), pp. 85-111; Rose et al., J. Biol. Chem. 270 (1995), pp. 26370- 26376; Baumann, J. Mol. Biol.242 (1994), pp. 244-251; Angkawidjaja, et al, FEBS Lett. 581(2007), pp. 5060-5064; Meier et al, J. Biol. Chem. 282 (2007), pp. 31477-31483;
Bauche et al, J. Biol. Chem. 281 (2006), pp. 16914-16926; Baumann et al, EMBO J. 12 (1993), pp. 3357-3364; Angkawidjaja et al., FEBS Lett. 579(2005), pp. 4707-4712).
[0036] The precipatable-beta roll tags and precipatable-beta roll cassettes described herein are class of designed peptides which possess the ability to reversibly precipitate in response to calcium ions. In one aspect, the invention described herein relates to the surprising finding that PBRCs (e.g. PBRTs repeats of sequence GGAGNDTLY (SEQ ID NO: 1)) undergo reversible precipitation upon calcium binding. In another aspect, the invention described herein relates to the surprising finding that attachment of a PBRC to a second molecule (e.g. attachment to a protein as a fusion protein comprising an in- frame) can be used to purify the second molecule through reversible precipitation. In another aspect, the invention described herein relates to the use of calcium concentration changes at room temperature to induce precipitation of recombinant molecules comprising a precipatable beta-roll tag.
[0037] In addition to target polypeptides, the PBRCs describe herein are also suitable for purifying non-peptide purification moieties of widely varying types, including, for example, lipids, oligonucleotides and carbohydrates, small organic or inorganic molecules, proteins, single-stranded or double-stranded oligonucleotides, polynucleotides. In certain aspects, applications for the methods and compositions described herein include, but are not limited to, the purification of recombinant proteins the removal of target proteins from a sample, and detection of compounds for diagnostic purposes. The invention also extends to the antibodies that specifically bind to a PBRT or a PBRC and the methods for using the PBRTs and PBRCs described herein.
[0038] Without wishing to be bound to theory, in certain embodiments, the PBRTs and PBRCs described herein can undergo a reversible Ca2+ binding dependent transition wherein they are structurally disordered and highly soluble in a medium below a Ca2+ concentration (or free Ca2+) transition concentration, but exhibit a disorder to order phase transition when the Ca2+ or free Ca2+ concentration is raised above the Ca2+ (or free Ca2+) transition concentration. Again, without wishing to be bound by theory, in some embodiments, the disorder to order phase transition leads to precipitation of the PBRTs or PBRCs. Precipitation of PBRC can be used to remove and isolated them from solution (e.g. by centrifugation). In one embodiment, the invention described herein relates to a PBRC which functions reversible Ca2+ precipatable tag when linked to a purification moiety of interest. In embodiments where the PBRC is linked to a purification moiety of interest, the methods described herein can be used to induce precipitation of the PBRC linked purification moiety. Because the transition concentration dependent phase transition is reversible, the PBRT and PBRC can be resolubilized in a medium having a Ca2+ concentration (or free Ca2+) below the transition concentration. In certain embodiments, this can be accomplished by introducing medium having reduced, or no Ca2+, or by removing, or chelating Ca2+ from the medium. When the precipitate is resuspended in calcium-free buffer or in a buffer comprising a calcium ion chelator (e.g. EGTA or EDTA), the precipitate resuspends into solution.
[0039] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.
[0040] The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%.
[0041] In certain embodiments, the term "precipatable-beta roll tag" (PBRT) refers to an amino acid sequence having the sequence GGAGNDTLY (SEQ ID NO: 1). In certain embodiments, a PBRT refers to an amino acid sequence having the amino acid sequence GXXXXXXXX (SEQ ID NO: 1343), wherein (a) the X at position 2 is an amino acid selected from the group consisting of glycine, asparagine or aspartic acid, and (b) the X at position 3 is an amino acid selected from the group consisting of alanine, serine, glycine, aspartic acid, glutamic acid, leucine or asparagine, and (c) the X at position 4 is an amino acid selected from the group consisting of glycine or alanine, and (d) the X at position 5 is an amino acid selected from the group consisting of asparagine, aspartic acid, alanine, or serine, and (e) the X at position 6 is an amino acid selected from the group consisting of aspartic acid or asparagine, (f) the X at position 7 is an amino acid selected from the group consisting of threonine, isoleucine, valine, or leucine, and (g) the X at position 8 is an amino acid selected from the group consisting of leucine, isoleucine, or phenylalanine, and (h) the X at position 9 is an amino acid selected from the group consisting of tyrosine, isoleucine, valine, phenylalanine, threonine, asparagine, aspartic acid, lysine or serine, or a nucleic acid encoding the same. A PBRT refers to an amino acid sequence having the sequence set forth in any of SEQ ID NOs: 25-1337. [0042] Table 1. SEQ ID NOs: 25-1337
Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO)
GDNASDLFS SEQ ID NO: 25 GGGGNDTLI SEQ ID NO: 463 GGAGDDTLV SEQ ID NO: 901
GNAGNDTLY SEQ ID NO: 26 GNGGNDTLI SEQ ID NO: 464 GNAGDDTLV SEQ ID NO: 902
GDAGNDTLY SEQ ID NO: 27 GDGGNDTLI SEQ ID NO: 465 GDAGDDTLV SEQ ID NO: 903
GGSGNDTLY SEQ ID NO: 28 GGDGNDTLI SEQ ID NO: 466 GGSGDDTLV SEQ ID NO: 904
GNSGNDTLY SEQ ID NO: 29 GNDGNDTLI SEQ ID NO: 467 GNSGDDTLV SEQ ID NO: 905
GDSGNDTLY SEQ ID NO: 30 GDDGNDTLI SEQ ID NO: 468 GDSGDDTLV SEQ ID NO: 906
GGGGNDTLY SEQ ID NO: 31 GGAGDDTLI SEQ ID NO: 469 GGGGDDTLV SEQ ID NO: 907
GNGGNDTLY SEQ ID NO: 32 GNAGDDTLI SEQ ID NO: 470 GNGGDDTLV SEQ ID NO: 908
GDGGNDTLY SEQ ID NO: 33 GDAGDDTLI SEQ ID NO: 471 GDGGDDTLV SEQ ID NO: 909
GGDGNDTLY SEQ ID NO: 34 GGSGDDTLI SEQ ID NO: 472 GGDGDDTLV SEQ ID NO: 910
GNDGNDTLY SEQ ID NO: 35 GNSGDDTLI SEQ ID NO: 473 GNDGDDTLV SEQ ID NO: 911
GDDGNDTLY SEQ ID NO: 36 GDSGDDTLI SEQ ID NO: 474 GDDGDDTLV SEQ ID NO: 912
GGAGDDTLY SEQ ID NO: 37 GGGGDDTLI SEQ ID NO: 475 GGAGADTLV SEQ ID NO: 913
GNAGDDTLY SEQ ID NO: 38 GNGGDDTLI SEQ ID NO: 476 GNAGADTLV SEQ ID NO: 914
GDAGDDTLY SEQ ID NO: 39 GDGGDDTLI SEQ ID NO: 477 GDAGADTLV SEQ ID NO: 915
GGSGDDTLY SEQ ID NO: 40 GGDGDDTLI SEQ ID NO: 478 GGSGADTLV SEQ ID NO: 916
GNSGDDTLY SEQ ID NO: 41 GNDGDDTLI SEQ ID NO: 479 GNSGADTLV SEQ ID NO: 917
GDSGDDTLY SEQ ID NO: 42 GDDGDDTLI SEQ ID NO: 480 GDSGADTLV SEQ ID NO: 918
GGGGDDTLY SEQ ID NO: 43 GGAGADTLI SEQ ID NO: 481 GGGGADTLV SEQ ID NO: 919
GNGGDDTLY SEQ ID NO: 44 GNAGADTLI SEQ ID NO: 482 GNGGADTLV SEQ ID NO: 920
GDGGDDTLY SEQ ID NO: 45 GDAGADTLI SEQ ID NO: 483 GDGGADTLV SEQ ID NO: 921
GGDGDDTLY SEQ ID NO: 46 GGSGADTLI SEQ ID NO: 484 GGDGADTLV SEQ ID NO: 922
GNDGDDTLY SEQ ID NO: 47 GNSGADTLI SEQ ID NO: 485 GNDGADTLV SEQ ID NO: 923
GDDGDDTLY SEQ ID NO: 48 GDSGADTLI SEQ ID NO: 486 GDDGADTLV SEQ ID NO: 924
GGAGADTLY SEQ ID NO: 49 GGGGADTLI SEQ ID NO: 487 GGAGNNTLV SEQ ID NO: 925
GNAGADTLY SEQ ID NO: 50 GNGGADTLI SEQ ID NO: 488 GNAGNNTLV SEQ ID NO: 926
GDAGADTLY SEQ ID NO: 51 GDGGADTLI SEQ ID NO: 489 GDAGNNTLV SEQ ID NO: 927
GGSGADTLY SEQ ID NO: 52 GGDGADTLI SEQ ID NO: 490 GGSGNNTLV SEQ ID NO: 928
GNSGADTLY SEQ ID NO: 53 GNDGADTLI SEQ ID NO: 491 GNSGNNTLV SEQ ID NO: 929
GDSGADTLY SEQ ID NO: 54 GDDGADTLI SEQ ID NO: 492 GDSGNNTLV SEQ ID NO: 930
GGGGADTLY SEQ ID NO: 55 GGAGNNTLI SEQ ID NO: 493 GGGGNNTLV SEQ ID NO: 931
GNGGADTLY SEQ ID NO: 56 GNAGNNTLI SEQ ID NO: 494 GNGGNNTLV SEQ ID NO: 932
GDGGADTLY SEQ ID NO: 57 GDAGNNTLI SEQ ID NO: 495 GDGGNNTLV SEQ ID NO: 933
GGDGADTLY SEQ ID NO: 58 GGSGNNTLI SEQ ID NO: 496 GGDGNNTLV SEQ ID NO: 934
GNDGADTLY SEQ ID NO: 59 GNSGNNTLI SEQ ID NO: 497 GNDGNNTLV SEQ ID NO: 935
GDDGADTLY SEQ ID NO: 60 GDSGNNTLI SEQ ID NO: 498 GDDGNNTLV SEQ ID NO: 936
GGAGNNTLY SEQ ID NO: 61 GGGGNNTLI SEQ ID NO: 499 GGAGDNTLV SEQ ID NO: 937
GNAGNNTLY SEQ ID NO: 62 GNGGNNTLI SEQ ID NO: 500 GNAGDNTLV SEQ ID NO: 938
GDAGNNTLY SEQ ID NO: 63 GDGGNNTLI SEQ ID NO: 501 GDAGDNTLV SEQ ID NO: 939
GGSGNNTLY SEQ ID NO: 64 GGDGNNTLI SEQ ID NO: 502 GGSGDNTLV SEQ ID NO: 940
GNSGNNTLY SEQ ID NO: 65 GNDGNNTLI SEQ ID NO: 503 GNSGDNTLV SEQ ID NO: 941
GDSGNNTLY SEQ ID NO: 66 GDDGNNTLI SEQ ID NO: 504 GDSGDNTLV SEQ ID NO: 942
GGGGNNTLY SEQ ID NO: 67 GGAGDNTLI SEQ ID NO: 505 GGGGDNTLV SEQ ID NO: 943
GNGGNNTLY SEQ ID NO: 68 GNAGDNTLI SEQ ID NO: 506 GNGGDNTLV SEQ ID NO: 944
GDGGNNTLY SEQ ID NO: 69 GDAGDNTLI SEQ ID NO: 507 GDGGDNTLV SEQ ID NO: 945
GGDGNNTLY SEQ ID NO: 70 GGSGDNTLI SEQ ID NO: 508 GGDGDNTLV SEQ ID NO: 946
GNDGNNTLY SEQ ID NO: 71 GNSGDNTLI SEQ ID NO: 509 GNDGDNTLV SEQ ID NO: 947
GDDGNNTLY SEQ ID NO: 72 GDSGDNTLI SEQ ID NO: 510 GDDGDNTLV SEQ ID NO: 948
GGAGDNTLY SEQ ID NO: 73 GGGGDNTLI SEQ ID NO: 511 GGAGANTLV SEQ ID NO: 949
GNAGDNTLY SEQ ID NO: 74 GNGGDNTLI SEQ ID NO: 512 GNAGANTLV SEQ ID NO: 950
GDAGDNTLY SEQ ID NO: 75 GDGGDNTLI SEQ ID NO: 513 GDAGANTLV SEQ ID NO: 951 Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO)
GGSGDNTLY SEQ ID NO 76 GGDGDNTLI SEQ ID NO: 514 GGSGANTLV SEQ ID NO: 952
GNSGDNTLY SEQ ID NO 77 GNDGDNTLI SEQ ID NO: 515 GNSGANTLV SEQ ID NO: 953
GDSGDNTLY SEQ ID NO 78 GDDGDNTLI SEQ ID NO: 516 GDSGANTLV SEQ ID NO: 954
GGGGDNTLY SEQ ID NO 79 GGAGANTLI SEQ ID NO: 517 GGGGANTLV SEQ ID NO: 955
GNGGDNTLY SEQ ID NO 80 GNAGANTLI SEQ ID NO: 518 GNGGANTLV SEQ ID NO: 956
GDGGDNTLY SEQ ID NO 81 GDAGANTLI SEQ ID NO: 519 GDGGANTLV SEQ ID NO: 957
GGDGDNTLY SEQ ID NO 82 GGSGANTLI SEQ ID NO: 520 GGDGANTLV SEQ ID NO: 958
GNDGDNTLY SEQ ID NO 83 GNSGANTLI SEQ ID NO: 521 GNDGANTLV SEQ ID NO: 959
GDDGDNTLY SEQ ID NO 84 GDSGANTLI SEQ ID NO: 522 GDDGANTLV SEQ ID NO: 960
GGAGANTLY SEQ ID NO 85 GGGGANTLI SEQ ID NO: 523 GGAGNDILV SEQ ID NO: 961
GNAGANTLY SEQ ID NO 86 GNGGANTLI SEQ ID NO: 524 GNAGNDILV SEQ ID NO: 962
GDAGANTLY SEQ ID NO 87 GDGGANTLI SEQ ID NO: 525 GDAGNDILV SEQ ID NO: 963
GGSGANTLY SEQ ID NO 88 GGDGANTLI SEQ ID NO: 526 GGSGNDILV SEQ ID NO: 964
GNSGANTLY SEQ ID NO 89 GNDGANTLI SEQ ID NO: 527 GNSGNDILV SEQ ID NO: 965
GDSGANTLY SEQ ID NO 90 GDDGANTLI SEQ ID NO: 528 GDSGNDILV SEQ ID NO: 966
GGGGANTLY SEQ ID NO 91 GGAGNDILI SEQ ID NO: 529 GGGGNDILV SEQ ID NO: 967
GNGGANTLY SEQ ID NO 92 GNAGNDILI SEQ ID NO: 530 GNGGNDILV SEQ ID NO: 968
GDGGANTLY SEQ ID NO 93 GDAGNDILI SEQ ID NO: 531 GDGGNDILV SEQ ID NO: 969
GGDGANTLY SEQ ID NO 94 GGSGNDILI SEQ ID NO: 532 GGDGNDILV SEQ ID NO: 970
GNDGANTLY SEQ ID NO 95 GNSGNDILI SEQ ID NO: 533 GNDGNDILV SEQ ID NO: 971
GDDGANTLY SEQ ID NO 96 GDSGNDILI SEQ ID NO: 534 GDDGNDILV SEQ ID NO: 972
GGAGNDILY SEQ ID NO 97 GGGGNDILI SEQ ID NO: 535 GGAGDDILV SEQ ID NO: 973
GNAGNDILY SEQ ID NO 98 GNGGNDILI SEQ ID NO: 536 GNAGDDILV SEQ ID NO: 974
GDAGNDILY SEQ ID NO 99 GDGGNDILI SEQ ID NO: 537 GDAGDDILV SEQ ID NO: 975
GGSGNDILY SEQ ID NO 100 GGDGNDILI SEQ ID NO: 538 GGSGDDILV SEQ ID NO: 976
GNSGNDILY SEQ ID NO 101 GNDGNDILI SEQ ID NO: 539 GNSGDDILV SEQ ID NO: 977
GDSGNDILY SEQ ID NO 102 GDDGNDILI SEQ ID NO: 540 GDSGDDILV SEQ ID NO: 978
GGGGNDILY SEQ ID NO 103 GGAGDDILI SEQ ID NO: 541 GGGGDDILV SEQ ID NO: 979
GNGGNDILY SEQ ID NO 104 GNAGDDILI SEQ ID NO: 542 GNGGDDILV SEQ ID NO: 980
GDGGNDILY SEQ ID NO 105 GDAGDDILI SEQ ID NO: 543 GDGGDDILV SEQ ID NO: 981
GGDGNDILY SEQ ID NO 106 GGSGDDILI SEQ ID NO: 544 GGDGDDILV SEQ ID NO: 982
GNDGNDILY SEQ ID NO 107 GNSGDDILI SEQ ID NO: 545 GNDGDDILV SEQ ID NO: 983
GDDGNDILY SEQ ID NO 108 GDSGDDILI SEQ ID NO: 546 GDDGDDILV SEQ ID NO: 984
GGAGDDILY SEQ ID NO 109 GGGGDDILI SEQ ID NO: 547 GGAGADILV SEQ ID NO: 985
GNAGDDILY SEQ ID NO 110 GNGGDDILI SEQ ID NO: 548 GNAGADILV SEQ ID NO: 986
GDAGDDILY SEQ ID NO 111 GDGGDDILI SEQ ID NO: 549 GDAGADILV SEQ ID NO: 987
GGSGDDILY SEQ ID NO 112 GGDGDDILI SEQ ID NO: 550 GGSGADILV SEQ ID NO: 988
GNSGDDILY SEQ ID NO 113 GNDGDDILI SEQ ID NO: 551 GNSGADILV SEQ ID NO: 989
GDSGDDILY SEQ ID NO 114 GDDGDDILI SEQ ID NO: 552 GDSGADILV SEQ ID NO: 990
GGGGDDILY SEQ ID NO 115 GGAGADILI SEQ ID NO: 553 GGGGADILV SEQ ID NO: 991
GNGGDDILY SEQ ID NO 116 GNAGADILI SEQ ID NO: 554 GNGGADILV SEQ ID NO: 992
GDGGDDILY SEQ ID NO 117 GDAGADILI SEQ ID NO: 555 GDGGADILV SEQ ID NO: 993
GGDGDDILY SEQ ID NO 118 GGSGADILI SEQ ID NO: 556 GGDGADILV SEQ ID NO: 994
GNDGDDILY SEQ ID NO 119 GNSGADILI SEQ ID NO: 557 GNDGADILV SEQ ID NO: 995
GDDGDDILY SEQ ID NO 120 GDSGADILI SEQ ID NO: 558 GDDGADILV SEQ ID NO: 996
GGAGADILY SEQ ID NO 121 GGGGADILI SEQ ID NO: 559 GGAGNNILV SEQ ID NO: 997
GNAGADILY SEQ ID NO 122 GNGGADILI SEQ ID NO: 560 GNAGNNILV SEQ ID NO: 998
GDAGADILY SEQ ID NO 123 GDGGADILI SEQ ID NO: 561 GDAGNNILV SEQ ID NO: 999
GGSGADILY SEQ ID NO 124 GGDGADILI SEQ ID NO: 562 GGSGNNILV SEQ ID NO: 1000
GNSGADILY SEQ ID NO 125 GNDGADILI SEQ ID NO: 563 GNSGNNILV SEQ ID NO: 1001
GDSGADILY SEQ ID NO 126 GDDGADILI SEQ ID NO: 564 GDSGNNILV SEQ ID NO: 1002
GGGGADILY SEQ ID NO 127 GGAGNNILI SEQ ID NO: 565 GGGGNNILV SEQ ID NO: 1003
GNGGADILY SEQ ID NO 128 GNAGNNILI SEQ ID NO: 566 GNGGNNILV SEQ ID NO: 1004 Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO)
GDGGADILY SEQ ID NO 129 GDAGNNILI SEQ ID NO 567 GDGGNNILV SEQ ID NO 1005
GGDGADILY SEQ ID NO 130 GGSGNNILI SEQ ID NO 568 GGDGNNILV SEQ ID NO 1006
GNDGADILY SEQ ID NO 131 GNSGNNILI SEQ ID NO 569 GNDGNNILV SEQ ID NO 1007
GDDGADILY SEQ ID NO 132 GDSGNNILI SEQ ID NO 570 GDDGNNILV SEQ ID NO 1008
GGAG NILY SEQ ID NO 133 GGGGNNILI SEQ ID NO 571 GGAGDNILV SEQ ID NO 1009
GNAG NILY SEQ ID NO 134 GNGGNNILI SEQ ID NO 572 GNAGDNILV SEQ ID NO 1010
GDAG NILY SEQ ID NO 135 GDGGNNILI SEQ ID NO 573 GDAGDNILV SEQ ID NO 1011
GGSG NILY SEQ ID NO 136 GGDGNNILI SEQ ID NO 574 GGSGDNILV SEQ ID NO 1012
GNSG NILY SEQ ID NO 137 GNDGNNILI SEQ ID NO 575 GNSGDNILV SEQ ID NO 1013
GDSG NILY SEQ ID NO 138 GDDGNNILI SEQ ID NO 576 GDSGDNILV SEQ ID NO 1014
GGGG NILY SEQ ID NO 139 GGAGDNILI SEQ ID NO 577 GGGGDNILV SEQ ID NO 1015
GNGG NILY SEQ ID NO 140 GNAGDNILI SEQ ID NO 578 GNGGDNILV SEQ ID NO 1016
GDGG NILY SEQ ID NO 141 GDAGDNILI SEQ ID NO 579 GDGGDNILV SEQ ID NO 1017
GGDG NILY SEQ ID NO 142 GGSGDNILI SEQ ID NO 580 GGDGDNILV SEQ ID NO 1018
GNDG NILY SEQ ID NO 143 GNSGDNILI SEQ ID NO 581 GNDGDNILV SEQ ID NO 1019
GDDG NILY SEQ ID NO 144 GDSGDNILI SEQ ID NO 582 GDDGDNILV SEQ ID NO 1020
GGAGDNILY SEQ ID NO 145 GGGGDNILI SEQ ID NO 583 GGAGANILV SEQ ID NO 1021
GNAGDNILY SEQ ID NO 146 GNGGDNILI SEQ ID NO 584 GNAGANILV SEQ ID NO 1022
GDAGDNILY SEQ ID NO 147 GDGGDNILI SEQ ID NO 585 GDAGANILV SEQ ID NO 1023
GGSGDNILY SEQ ID NO 148 GGDGDNILI SEQ ID NO 586 GGSGANILV SEQ ID NO 1024
GNSGDNILY SEQ ID NO 149 GNDGDNILI SEQ ID NO 587 GNSGANILV SEQ ID NO 1025
GDSGDNILY SEQ ID NO 150 GDDGDNILI SEQ ID NO 588 GDSGANILV SEQ ID NO 1026
GGGGDNILY SEQ ID NO 151 GGAGANILI SEQ ID NO 589 GGGGANILV SEQ ID NO 1027
GNGGDNILY SEQ ID NO 152 GNAGANILI SEQ ID NO 590 GNGGANILV SEQ ID NO 1028
GDGGDNILY SEQ ID NO 153 GDAGANILI SEQ ID NO 591 GDGGANILV SEQ ID NO 1029
GGDGDNILY SEQ ID NO 154 GGSGANILI SEQ ID NO 592 GGDGANILV SEQ ID NO 1030
GNDGDNILY SEQ ID NO 155 GNSGANILI SEQ ID NO 593 GNDGANILV SEQ ID NO 1031
GDDGDNILY SEQ ID NO 156 GDSGANILI SEQ ID NO 594 GDDGANILV SEQ ID NO 1032
GGAGANILY SEQ ID NO 157 GGGGANILI SEQ ID NO 595 GGAGNDVLV SEQ ID NO 1033
GNAGANILY SEQ ID NO 158 GNGGANILI SEQ ID NO 596 GNAGNDVLV SEQ ID NO 1034
GDAGANILY SEQ ID NO 159 GDGGANILI SEQ ID NO 597 GDAGNDVLV SEQ ID NO 1035
GGSGANILY SEQ ID NO 160 GGDGANILI SEQ ID NO 598 GGSGNDVLV SEQ ID NO 1036
GNSGANILY SEQ ID NO 161 GNDGANILI SEQ ID NO 599 GNSGNDVLV SEQ ID NO 1037
GDSGANILY SEQ ID NO 162 GDDGANILI SEQ ID NO 600 GDSGNDVLV SEQ ID NO 1038
GGGGANILY SEQ ID NO 163 GGAGNDVLI SEQ ID NO 601 GGGGNDVLV SEQ ID NO 1039
GNGGANILY SEQ ID NO 164 GNAGNDVLI SEQ ID NO 602 GNGGNDVLV SEQ ID NO 1040
GDGGANILY SEQ ID NO 165 GDAGNDVLI SEQ ID NO 603 GDGGNDVLV SEQ ID NO 1041
GGDGANILY SEQ ID NO 166 GGSGNDVLI SEQ ID NO 604 GGDGNDVLV SEQ ID NO 1042
GNDGANILY SEQ ID NO 167 GNSGNDVLI SEQ ID NO 605 GNDGNDVLV SEQ ID NO 1043
GDDGANILY SEQ ID NO 168 GDSGNDVLI SEQ ID NO 606 GDDGNDVLV SEQ ID NO 1044
GGAGNDVLY SEQ ID NO 169 GGGGNDVLI SEQ ID NO 607 GGAGDDVLV SEQ ID NO 1045
GNAGNDVLY SEQ ID NO 170 GNGGNDVLI SEQ ID NO 608 GNAGDDVLV SEQ ID NO 1046
GDAGNDVLY SEQ ID NO 171 GDGGNDVLI SEQ ID NO 609 GDAGDDVLV SEQ ID NO 1047
GGSGNDVLY SEQ ID NO 172 GGDGNDVLI SEQ ID NO 610 GGSGDDVLV SEQ ID NO 1048
GNSGNDVLY SEQ ID NO 173 GNDGNDVLI SEQ ID NO 611 GNSGDDVLV SEQ ID NO 1049
GDSGNDVLY SEQ ID NO 174 GDDGNDVLI SEQ ID NO 612 GDSGDDVLV SEQ ID NO 1050
GGGGNDVLY SEQ ID NO 175 GGAGDDVLI SEQ ID NO 613 GGGGDDVLV SEQ ID NO 1051
GNGGNDVLY SEQ ID NO 176 GNAGDDVLI SEQ ID NO 614 GNGGDDVLV SEQ ID NO 1052
GDGGNDVLY SEQ ID NO 177 GDAGDDVLI SEQ ID NO 615 GDGGDDVLV SEQ ID NO 1053
GGDGNDVLY SEQ ID NO 178 GGSGDDVLI SEQ ID NO 616 GGDGDDVLV SEQ ID NO 1054
GNDGNDVLY SEQ ID NO 179 GNSGDDVLI SEQ ID NO 617 GNDGDDVLV SEQ ID NO 1055
GDDGNDVLY SEQ ID NO 180 GDSGDDVLI SEQ ID NO 618 GDDGDDVLV SEQ ID NO 1056
GGAGDDVLY SEQ ID NO 181 GGGGDDVLI SEQ ID NO 619 GGAGADVLV SEQ ID NO 1057 Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO)
GNAGDDVLY SEQ ID NO: 182 GNGGDDVLI SEQ ID NO 620 GNAGADVLV SEQ ID NO 1058
GDAGDDVLY SEQ ID NO: 183 GDGGDDVLI SEQ ID NO 621 GDAGADVLV SEQ ID NO 1059
GGSGDDVLY SEQ ID NO: 184 GGDGDDVLI SEQ ID NO 622 GGSGADVLV SEQ ID NO 1060
GNSGDDVLY SEQ ID NO: 185 GNDGDDVLI SEQ ID NO 623 GNSGADVLV SEQ ID NO 1061
GDSGDDVLY SEQ ID NO: 186 GDDGDDVLI SEQ ID NO 624 GDSGADVLV SEQ ID NO 1062
GGGGDDVLY SEQ ID NO: 187 GGAGADVLI SEQ ID NO 625 GGGGADVLV SEQ ID NO 1063
GNGGDDVLY SEQ ID NO: 188 GNAGADVLI SEQ ID NO 626 GNGGADVLV SEQ ID NO 1064
GDGGDDVLY SEQ ID NO: 189 GDAGADVLI SEQ ID NO 627 GDGGADVLV SEQ ID NO 1065
GGDGDDVLY SEQ ID NO: 190 GGSGADVLI SEQ ID NO 628 GGDGADVLV SEQ ID NO 1066
GNDGDDVLY SEQ ID NO: 191 GNSGADVLI SEQ ID NO 629 GNDGADVLV SEQ ID NO 1067
GDDGDDVLY SEQ ID NO: 192 GDSGADVLI SEQ ID NO 630 GDDGADVLV SEQ ID NO 1068
GGAGADVLY SEQ ID NO: 193 GGGGADVLI SEQ ID NO 631 GGAGNNVLV SEQ ID NO 1069
GNAGADVLY SEQ ID NO: 194 GNGGADVLI SEQ ID NO 632 GNAGNNVLV SEQ ID NO 1070
GDAGADVLY SEQ ID NO: 195 GDGGADVLI SEQ ID NO 633 GDAGNNVLV SEQ ID NO 1071
GGSGADVLY SEQ ID NO: 196 GGDGADVLI SEQ ID NO 634 GGSGNNVLV SEQ ID NO 1072
GNSGADVLY SEQ ID NO: 197 GNDGADVLI SEQ ID NO 635 GNSGNNVLV SEQ ID NO 1073
GDSGADVLY SEQ ID NO: 198 GDDGADVLI SEQ ID NO 636 GDSGNNVLV SEQ ID NO 1074
GGGGADVLY SEQ ID NO: 199 GGAGNNVLI SEQ ID NO 637 GGGGNNVLV SEQ ID NO 1075
GNGGADVLY SEQ ID NO: 200 GNAGNNVLI SEQ ID NO 638 GNGGNNVLV SEQ ID NO 1076
GDGGADVLY SEQ ID NO: 201 GDAGNNVLI SEQ ID NO 639 GDGGNNVLV SEQ ID NO 1077
GGDGADVLY SEQ ID NO: 202 GGSGNNVLI SEQ ID NO 640 GGDGNNVLV SEQ ID NO 1078
GNDGADVLY SEQ ID NO: 203 GNSGNNVLI SEQ ID NO 641 GNDGNNVLV SEQ ID NO 1079
GDDGADVLY SEQ ID NO: 204 GDSGNNVLI SEQ ID NO 642 GDDGNNVLV SEQ ID NO 1080
GGAG VLY SEQ ID NO: 205 GGGGNNVLI SEQ ID NO 643 GGAGDNVLV SEQ ID NO 1081
GNAG VLY SEQ ID NO: 206 GNGGNNVLI SEQ ID NO 644 GNAGDNVLV SEQ ID NO 1082
GDAG VLY SEQ ID NO: 207 GDGGNNVLI SEQ ID NO 645 GDAGDNVLV SEQ ID NO 1083
GGSG NVLY SEQ ID NO: 208 GGDGNNVLI SEQ ID NO 646 GGSGDNVLV SEQ ID NO 1084
GNSG NVLY SEQ ID NO: 209 GNDGNNVLI SEQ ID NO 647 GNSGDNVLV SEQ ID NO 1085
GDSG NVLY SEQ ID NO: 210 GDDGNNVLI SEQ ID NO 648 GDSGDNVLV SEQ ID NO 1086
GGGG VLY SEQ ID NO: 211 GGAGDNVLI SEQ ID NO 649 GGGGDNVLV SEQ ID NO 1087
GNGG VLY SEQ ID NO: 212 GNAGDNVLI SEQ ID NO 650 GNGGDNVLV SEQ ID NO 1088
GDGG VLY SEQ ID NO: 213 GDAGDNVLI SEQ ID NO 651 GDGGDNVLV SEQ ID NO 1089
GGDG VLY SEQ ID NO: 214 GGSGDNVLI SEQ ID NO 652 GGDGDNVLV SEQ ID NO 1090
GNDG VLY SEQ ID NO: 215 GNSGDNVLI SEQ ID NO 653 GNDGDNVLV SEQ ID NO 1091
GDDG VLY SEQ ID NO: 216 GDSGDNVLI SEQ ID NO 654 GDDGDNVLV SEQ ID NO 1092
GGAGD VLY SEQ ID NO: 217 GGGGDNVLI SEQ ID NO 655 GGAGANVLV SEQ ID NO 1093
GNAGD VLY SEQ ID NO: 218 GNGGDNVLI SEQ ID NO 656 GNAGANVLV SEQ ID NO 1094
GDAGD VLY SEQ ID NO: 219 GDGGDNVLI SEQ ID NO 657 GDAGANVLV SEQ ID NO 1095
GGSGDNVLY SEQ ID NO: 220 GGDGDNVLI SEQ ID NO 658 GGSGANVLV SEQ ID NO 1096
GNSGDNVLY SEQ ID NO: 221 GNDGDNVLI SEQ ID NO 659 GNSGANVLV SEQ ID NO 1097
GDSGDNVLY SEQ ID NO: 222 GDDGDNVLI SEQ ID NO 660 GDSGANVLV SEQ ID NO 1098
GGGGD VLY SEQ ID NO: 223 GGAGANVLI SEQ ID NO 661 GGGGANVLV SEQ ID NO 1099
GNGGD VLY SEQ ID NO: 224 GNAGANVLI SEQ ID NO 662 GNGGANVLV SEQ ID NO 1100
GDGGD VLY SEQ ID NO: 225 GDAGANVLI SEQ ID NO 663 GDGGANVLV SEQ ID NO 1101
GGDGD VLY SEQ ID NO: 226 GGSGANVLI SEQ ID NO 664 GGDGANVLV SEQ ID NO 1102
GNDGD VLY SEQ ID NO: 227 GNSGANVLI SEQ ID NO 665 GNDGANVLV SEQ ID NO 1103
GDDGD VLY SEQ ID NO: 228 GDSGANVLI SEQ ID NO 666 GDDGANVLV SEQ ID NO 1104
GGAGANVLY SEQ ID NO: 229 GGGGANVLI SEQ ID NO 667 GGAGNDTIV SEQ ID NO 1105
GNAGA VLY SEQ ID NO: 230 GNGGANVLI SEQ ID NO 668 GNAGNDTIV SEQ ID NO 1106
GDAGA VLY SEQ ID NO: 231 GDGGANVLI SEQ ID NO 669 GDAGNDTIV SEQ ID NO 1107
GGSGANVLY SEQ ID NO: 232 GGDGANVLI SEQ ID NO 670 GGSGNDTIV SEQ ID NO 1108
GNSGANVLY SEQ ID NO: 233 GNDGANVLI SEQ ID NO 671 GNSGNDTIV SEQ ID NO 1109
GDSGANVLY SEQ ID NO: 234 GDDGANVLI SEQ ID NO 672 GDSGNDTIV SEQ ID NO 1110 Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO)
GGGGANVLY SEQ ID NO 235 GGAGNDTII SEQ ID NO: 673 GGGGNDTIV SEQ ID NO 1111
GNGGANVLY SEQ ID NO 236 GNAGNDTII SEQ ID NO: 674 GNGGNDTIV SEQ ID NO 1112
GDGGANVLY SEQ ID NO 237 GDAGNDTII SEQ ID NO: 675 GDGGNDTIV SEQ ID NO 1113
GGDGANVLY SEQ ID NO 238 GGSGNDTII SEQ ID NO: 676 GGDGNDTIV SEQ ID NO 1114
GNDGANVLY SEQ ID NO 239 GNSGNDTII SEQ ID NO: 677 GNDGNDTIV SEQ ID NO 1115
GDDGANVLY SEQ ID NO 240 GDSGNDTII SEQ ID NO: 678 GDDGNDTIV SEQ ID NO 1116
GGAGNDTIY SEQ ID NO 241 GGGGNDTII SEQ ID NO: 679 GGAGDDTIV SEQ ID NO 1117
GNAGNDTIY SEQ ID NO 242 GNGGNDTII SEQ ID NO: 680 GNAGDDTIV SEQ ID NO 1118
GDAGNDTIY SEQ ID NO 243 GDGGNDTII SEQ ID NO: 681 GDAGDDTIV SEQ ID NO 1119
GGSGNDTIY SEQ ID NO 244 GGDGNDTII SEQ ID NO: 682 GGSGDDTIV SEQ ID NO 1120
GNSGNDTIY SEQ ID NO 245 GNDGNDTII SEQ ID NO: 683 GNSGDDTIV SEQ ID NO 1121
GDSGNDTIY SEQ ID NO 246 GDDGNDTII SEQ ID NO: 684 GDSGDDTIV SEQ ID NO 1122
GGGGNDTIY SEQ ID NO 247 GGAGDDTII SEQ ID NO: 685 GGGGDDTIV SEQ ID NO 1123
GNGGNDTIY SEQ ID NO 248 GNAGDDTII SEQ ID NO: 686 GNGGDDTIV SEQ ID NO 1124
GDGGNDTIY SEQ ID NO 249 GDAGDDTII SEQ ID NO: 687 GDGGDDTIV SEQ ID NO 1125
GGDGNDTIY SEQ ID NO 250 GGSGDDTII SEQ ID NO: 688 GGDGDDTIV SEQ ID NO 1126
GNDGNDTIY SEQ ID NO 251 GNSGDDTII SEQ ID NO: 689 GNDGDDTIV SEQ ID NO 1127
GDDGNDTIY SEQ ID NO 252 GDSGDDTII SEQ ID NO: 690 GDDGDDTIV SEQ ID NO 1128
GGAGDDTIY SEQ ID NO 253 GGGGDDTII SEQ ID NO: 691 GGAGADTIV SEQ ID NO 1129
GNAGDDTIY SEQ ID NO 254 GNGGDDTII SEQ ID NO: 692 GNAGADTIV SEQ ID NO 1130
GDAGDDTIY SEQ ID NO 255 GDGGDDTII SEQ ID NO: 693 GDAGADTIV SEQ ID NO 1131
GGSGDDTIY SEQ ID NO 256 GGDGDDTII SEQ ID NO: 694 GGSGADTIV SEQ ID NO 1132
GNSGDDTIY SEQ ID NO 257 GNDGDDTII SEQ ID NO: 695 GNSGADTIV SEQ ID NO 1133
GDSGDDTIY SEQ ID NO 258 GDDGDDTII SEQ ID NO: 696 GDSGADTIV SEQ ID NO 1134
GGGGDDTIY SEQ ID NO 259 GGAGADTII SEQ ID NO: 697 GGGGADTIV SEQ ID NO 1135
GNGGDDTIY SEQ ID NO 260 GNAGADTII SEQ ID NO: 698 GNGGADTIV SEQ ID NO 1136
GDGGDDTIY SEQ ID NO 261 GDAGADTII SEQ ID NO: 699 GDGGADTIV SEQ ID NO 1137
GGDGDDTIY SEQ ID NO 262 GGSGADTII SEQ ID NO: 700 GGDGADTIV SEQ ID NO 1138
GNDGDDTIY SEQ ID NO 263 GNSGADTII SEQ ID NO: 701 GNDGADTIV SEQ ID NO 1139
GDDGDDTIY SEQ ID NO 264 GDSGADTII SEQ ID NO: 702 GDDGADTIV SEQ ID NO 1140
GGAGADTIY SEQ ID NO 265 GGGGADTII SEQ ID NO: 703 GGAGNNTIV SEQ ID NO 1141
GNAGADTIY SEQ ID NO 266 GNGGADTII SEQ ID NO: 704 GNAGNNTIV SEQ ID NO 1142
GDAGADTIY SEQ ID NO 267 GDGGADTII SEQ ID NO: 705 GDAGNNTIV SEQ ID NO 1143
GGSGADTIY SEQ ID NO 268 GGDGADTII SEQ ID NO: 706 GGSGNNTIV SEQ ID NO 1144
GNSGADTIY SEQ ID NO 269 GNDGADTII SEQ ID NO: 707 GNSGNNTIV SEQ ID NO 1145
GDSGADTIY SEQ ID NO 270 GDDGADTII SEQ ID NO: 708 GDSGNNTIV SEQ ID NO 1146
GGGGADTIY SEQ ID NO 271 GGAGNNTII SEQ ID NO: 709 GGGGNNTIV SEQ ID NO 1147
GNGGADTIY SEQ ID NO 272 GNAGNNTII SEQ ID NO: 710 GNGGNNTIV SEQ ID NO 1148
GDGGADTIY SEQ ID NO 273 GDAGNNTII SEQ ID NO: 711 GDGGNNTIV SEQ ID NO 1149
GGDGADTIY SEQ ID NO 274 GGSGNNTII SEQ ID NO: 712 GGDGNNTIV SEQ ID NO 1150
GNDGADTIY SEQ ID NO 275 GNSGNNTII SEQ ID NO: 713 GNDGNNTIV SEQ ID NO 1151
GDDGADTIY SEQ ID NO 276 GDSGNNTII SEQ ID NO: 714 GDDGNNTIV SEQ ID NO 1152
GGAGNNTIY SEQ ID NO 277 GGGGNNTII SEQ ID NO: 715 GGAGDNTIV SEQ ID NO 1153
GNAGNNTIY SEQ ID NO 278 GNGGNNTII SEQ ID NO: 716 GNAGDNTIV SEQ ID NO 1154
GDAGNNTIY SEQ ID NO 279 GDGGNNTII SEQ ID NO: 717 GDAGDNTIV SEQ ID NO 1155
GGSGNNTIY SEQ ID NO 280 GGDGNNTII SEQ ID NO: 718 GGSGDNTIV SEQ ID NO 1156
GNSGNNTIY SEQ ID NO 281 GNDGNNTII SEQ ID NO: 719 GNSGDNTIV SEQ ID NO 1157
GDSGNNTIY SEQ ID NO 282 GDDGNNTII SEQ ID NO: 720 GDSGDNTIV SEQ ID NO 1158
GGGGNNTIY SEQ ID NO 283 GGAGDNTII SEQ ID NO: 721 GGGGDNTIV SEQ ID NO 1159
GNGGNNTIY SEQ ID NO 284 GNAGDNTII SEQ ID NO: 722 GNGGDNTIV SEQ ID NO 1160
GDGGNNTIY SEQ ID NO 285 GDAGDNTII SEQ ID NO: 723 GDGGDNTIV SEQ ID NO 1161
GGDGNNTIY SEQ ID NO 286 GGSGDNTII SEQ ID NO: 724 GGDGDNTIV SEQ ID NO 1162
GNDGNNTIY SEQ ID NO 287 GNSGDNTII SEQ ID NO: 725 GNDGDNTIV SEQ ID NO 1163 Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO)
GDDGNNTIY SEQ ID NO 288 GDSGDNTII SEQ ID NO: 726 GDDGDNTIV SEQ ID NO 1164
GGAGDNTIY SEQ ID NO 289 GGGGDNTII SEQ ID NO: 727 GGAGANTIV SEQ ID NO 1165
GNAGDNTIY SEQ ID NO 290 GNGGDNTII SEQ ID NO: 728 GNAGANTIV SEQ ID NO 1166
GDAGDNTIY SEQ ID NO 291 GDGGDNTII SEQ ID NO: 729 GDAGANTIV SEQ ID NO 1167
GGSGDNTIY SEQ ID NO 292 GGDGDNTII SEQ ID NO: 730 GGSGANTIV SEQ ID NO 1168
GNSGDNTIY SEQ ID NO 293 GNDGDNTII SEQ ID NO: 731 GNSGANTIV SEQ ID NO 1169
GDSGDNTIY SEQ ID NO 294 GDDGDNTII SEQ ID NO: 732 GDSGANTIV SEQ ID NO 1170
GGGGDNTIY SEQ ID NO 295 GGAGANTII SEQ ID NO: 733 GGGGANTIV SEQ ID NO 1171
GNGGDNTIY SEQ ID NO 296 GNAGANTII SEQ ID NO: 734 GNGGANTIV SEQ ID NO 1172
GDGGDNTIY SEQ ID NO 297 GDAGANTII SEQ ID NO: 735 GDGGANTIV SEQ ID NO 1173
GGDGDNTIY SEQ ID NO 298 GGSGANTII SEQ ID NO: 736 GGDGANTIV SEQ ID NO 1174
GNDGDNTIY SEQ ID NO 299 GNSGANTII SEQ ID NO: 737 GNDGANTIV SEQ ID NO 1175
GDDGDNTIY SEQ ID NO 300 GDSGANTII SEQ ID NO: 738 GDDGANTIV SEQ ID NO 1176
GGAGANTIY SEQ ID NO 301 GGGGANTII SEQ ID NO: 739 GGAGNDIIV SEQ ID NO 1177
GNAGANTIY SEQ ID NO 302 GNGGANTII SEQ ID NO: 740 GNAGNDIIV SEQ ID NO 1178
GDAGANTIY SEQ ID NO 303 GDGGANTII SEQ ID NO: 741 GDAGNDIIV SEQ ID NO 1179
GGSGANTIY SEQ ID NO 304 GGDGANTII SEQ ID NO: 742 GGSGNDIIV SEQ ID NO 1180
GNSGANTIY SEQ ID NO 305 GNDGANTII SEQ ID NO: 743 GNSGNDIIV SEQ ID NO 1181
GDSGANTIY SEQ ID NO 306 GDDGANTII SEQ ID NO: 744 GDSGNDIIV SEQ ID NO 1182
GGGGANTIY SEQ ID NO 307 GGAGNDIII SEQ ID NO: 745 GGGGNDIIV SEQ ID NO 1183
GNGGANTIY SEQ ID NO 308 GNAGNDIII SEQ ID NO: 746 GNGGNDIIV SEQ ID NO 1184
GDGGANTIY SEQ ID NO 309 GDAGNDIII SEQ ID NO: 747 GDGGNDIIV SEQ ID NO 1185
GGDGANTIY SEQ ID NO 310 GGSGNDIII SEQ ID NO: 748 GGDGNDIIV SEQ ID NO 1186
GNDGANTIY SEQ ID NO 311 GNSGNDIII SEQ ID NO: 749 GNDGNDIIV SEQ ID NO 1187
GDDGANTIY SEQ ID NO 312 GDSGNDIII SEQ ID NO: 750 GDDGNDIIV SEQ ID NO 1188
GGAGNDIIY SEQ ID NO 313 GGGGNDIII SEQ ID NO: 751 GGAGDDIIV SEQ ID NO 1189
GNAGNDIIY SEQ ID NO 314 GNGGNDIII SEQ ID NO: 752 GNAGDDIIV SEQ ID NO 1190
GDAGNDIIY SEQ ID NO 315 GDGGNDIII SEQ ID NO: 753 GDAGDDIIV SEQ ID NO 1191
GGSGNDIIY SEQ ID NO 316 GGDGNDIII SEQ ID NO: 754 GGSGDDIIV SEQ ID NO 1192
GNSGNDIIY SEQ ID NO 317 GNDGNDIII SEQ ID NO: 755 GNSGDDIIV SEQ ID NO 1193
GDSGNDIIY SEQ ID NO 318 GDDGNDIII SEQ ID NO: 756 GDSGDDIIV SEQ ID NO 1194
GGGGNDIIY SEQ ID NO 319 GGAGDDIII SEQ ID NO: 757 GGGGDDIIV SEQ ID NO 1195
GNGGNDIIY SEQ ID NO 320 GNAGDDIII SEQ ID NO: 758 GNGGDDIIV SEQ ID NO 1196
GDGGNDIIY SEQ ID NO 321 GDAGDDIII SEQ ID NO: 759 GDGGDDIIV SEQ ID NO 1197
GGDGNDIIY SEQ ID NO 322 GGSGDDIII SEQ ID NO: 760 GGDGDDIIV SEQ ID NO 1198
GNDGNDIIY SEQ ID NO 323 GNSGDDIII SEQ ID NO: 761 GNDGDDIIV SEQ ID NO 1199
GDDGNDIIY SEQ ID NO 324 GDSGDDIII SEQ ID NO: 762 GDDGDDIIV SEQ ID NO 1200
GGAGDDIIY SEQ ID NO 325 GGGGDDIII SEQ ID NO: 763 GGAGADIIV SEQ ID NO 1201
GNAGDDIIY SEQ ID NO 326 GNGGDDIII SEQ ID NO: 764 GNAGADIIV SEQ ID NO 1202
GDAGDDIIY SEQ ID NO 327 GDGGDDIII SEQ ID NO: 765 GDAGADIIV SEQ ID NO 1203
GGSGDDIIY SEQ ID NO 328 GGDGDDIII SEQ ID NO: 766 GGSGADIIV SEQ ID NO 1204
GNSGDDIIY SEQ ID NO 329 GNDGDDIII SEQ ID NO: 767 GNSGADIIV SEQ ID NO 1205
GDSGDDIIY SEQ ID NO 330 GDDGDDIII SEQ ID NO: 768 GDSGADIIV SEQ ID NO 1206
GGGGDDIIY SEQ ID NO 331 GGAGADIII SEQ ID NO: 769 GGGGADIIV SEQ ID NO 1207
GNGGDDIIY SEQ ID NO 332 GNAGADIII SEQ ID NO: 770 GNGGADIIV SEQ ID NO 1208
GDGGDDIIY SEQ ID NO 333 GDAGADIII SEQ ID NO: 771 GDGGADIIV SEQ ID NO 1209
GGDGDDIIY SEQ ID NO 334 GGSGADIII SEQ ID NO: 772 GGDGADIIV SEQ ID NO 1210
GNDGDDIIY SEQ ID NO 335 GNSGADIII SEQ ID NO: 773 GNDGADIIV SEQ ID NO 1211
GDDGDDIIY SEQ ID NO 336 GDSGADIII SEQ ID NO: 774 GDDGADIIV SEQ ID NO 1212
GGAGADIIY SEQ ID NO 337 GGGGADIII SEQ ID NO: 775 GGAGNNIIV SEQ ID NO 1213
GNAGADIIY SEQ ID NO 338 GNGGADIII SEQ ID NO: 776 GNAGNNIIV SEQ ID NO 1214
GDAGADIIY SEQ ID NO 339 GDGGADIII SEQ ID NO: 777 GDAGNNIIV SEQ ID NO 1215
GGSGADIIY SEQ ID NO 340 GGDGADIII SEQ ID NO: 778 GGSGNNIIV SEQ ID NO 1216 Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO)
GNSGADIIY SEQ ID NO 341 GNDGADIII SEQ ID NO: 779 GNSGNNIIV SEQ ID NO 1217
GDSGADIIY SEQ ID NO 342 GDDGADIII SEQ ID NO: 780 GDSGNNIIV SEQ ID NO 1218
GGGGADIIY SEQ ID NO 343 GGAGNNIII SEQ ID NO: 781 GGGGNNIIV SEQ ID NO 1219
GNGGADIIY SEQ ID NO 344 GNAGNNIII SEQ ID NO: 782 GNGGNNIIV SEQ ID NO 1220
GDGGADIIY SEQ ID NO 345 GDAGNNIII SEQ ID NO: 783 GDGGNNIIV SEQ ID NO 1221
GGDGADIIY SEQ ID NO 346 GGSGNNIII SEQ ID NO: 784 GGDGNNIIV SEQ ID NO 1222
GNDGADIIY SEQ ID NO 347 GNSGNNIII SEQ ID NO: 785 GNDGNNIIV SEQ ID NO 1223
GDDGADIIY SEQ ID NO 348 GDSGNNIII SEQ ID NO: 786 GDDGNNIIV SEQ ID NO 1224
GGAG NIIY SEQ ID NO 349 GGGGNNIII SEQ ID NO: 787 GGAGDNIIV SEQ ID NO 1225
GNAG NIIY SEQ ID NO 350 GNGGNNIII SEQ ID NO: 788 GNAGDNIIV SEQ ID NO 1226
GDAG NIIY SEQ ID NO 351 GDGGNNIII SEQ ID NO: 789 GDAGDNIIV SEQ ID NO 1227
GGSG NIIY SEQ ID NO 352 GGDGNNIII SEQ ID NO: 790 GGSGDNIIV SEQ ID NO 1228
GNSG NIIY SEQ ID NO 353 GNDGNNIII SEQ ID NO: 791 GNSGDNIIV SEQ ID NO 1229
GDSG NIIY SEQ ID NO 354 GDDGNNIII SEQ ID NO: 792 GDSGDNIIV SEQ ID NO 1230
GGGG NIIY SEQ ID NO 355 GGAGDNIII SEQ ID NO: 793 GGGGDNIIV SEQ ID NO 1231
GNGG NIIY SEQ ID NO 356 GNAGDNIII SEQ ID NO: 794 GNGGDNIIV SEQ ID NO 1232
GDGG NIIY SEQ ID NO 357 GDAGDNIII SEQ ID NO: 795 GDGGDNIIV SEQ ID NO 1233
GGDG NIIY SEQ ID NO 358 GGSGDNIII SEQ ID NO: 796 GGDGDNIIV SEQ ID NO 1234
GNDG NIIY SEQ ID NO 359 GNSGDNIII SEQ ID NO: 797 GNDGDNIIV SEQ ID NO 1235
GDDG NIIY SEQ ID NO 360 GDSGDNIII SEQ ID NO: 798 GDDGDNIIV SEQ ID NO 1236
GGAGDNIIY SEQ ID NO 361 GGGGDNIII SEQ ID NO: 799 GGAGANIIV SEQ ID NO 1237
GNAGDNIIY SEQ ID NO 362 GNGGDNIII SEQ ID NO: 800 GNAGANIIV SEQ ID NO 1238
GDAGDNIIY SEQ ID NO 363 GDGGDNIII SEQ ID NO: 801 GDAGANIIV SEQ ID NO 1239
GGSGDNIIY SEQ ID NO 364 GGDGDNIII SEQ ID NO: 802 GGSGANIIV SEQ ID NO 1240
GNSGDNIIY SEQ ID NO 365 GNDGDNIII SEQ ID NO: 803 GNSGANIIV SEQ ID NO 1241
GDSGDNIIY SEQ ID NO 366 GDDGDNIII SEQ ID NO: 804 GDSGANIIV SEQ ID NO 1242
GGGGDNIIY SEQ ID NO 367 GGAGANIII SEQ ID NO: 805 GGGGANIIV SEQ ID NO 1243
GNGGDNIIY SEQ ID NO 368 GNAGANIII SEQ ID NO: 806 GNGGANIIV SEQ ID NO 1244
GDGGDNIIY SEQ ID NO 369 GDAGANIII SEQ ID NO: 807 GDGGANIIV SEQ ID NO 1245
GGDGDNIIY SEQ ID NO 370 GGSGANIII SEQ ID NO: 808 GGDGANIIV SEQ ID NO 1246
GNDGDNIIY SEQ ID NO 371 GNSGANIII SEQ ID NO: 809 GNDGANIIV SEQ ID NO 1247
GDDGDNIIY SEQ ID NO 372 GDSGANIII SEQ ID NO: 810 GDDGANIIV SEQ ID NO 1248
GGAGANIIY SEQ ID NO 373 GGGGANIII SEQ ID NO: 811 GGAGNDVIV SEQ ID NO 1249
GNAGANIIY SEQ ID NO 374 GNGGANIII SEQ ID NO: 812 GNAGNDVIV SEQ ID NO 1250
GDAGANIIY SEQ ID NO 375 GDGGANIII SEQ ID NO: 813 GDAGNDVIV SEQ ID NO 1251
GGSGANIIY SEQ ID NO 376 GGDGANIII SEQ ID NO: 814 GGSGNDVIV SEQ ID NO 1252
GNSGANIIY SEQ ID NO 377 GNDGANIII SEQ ID NO: 815 GNSGNDVIV SEQ ID NO 1253
GDSGANIIY SEQ ID NO 378 GDDGANIII SEQ ID NO: 816 GDSGNDVIV SEQ ID NO 1254
GGGGANIIY SEQ ID NO 379 GGAGNDVII SEQ ID NO: 817 GGGGNDVIV SEQ ID NO 1255
GNGGANIIY SEQ ID NO 380 GNAGNDVII SEQ ID NO: 818 GNGGNDVIV SEQ ID NO 1256
GDGGANIIY SEQ ID NO 381 GDAGNDVII SEQ ID NO: 819 GDGGNDVIV SEQ ID NO 1257
GGDGANIIY SEQ ID NO 382 GGSGNDVII SEQ ID NO: 820 GGDGNDVIV SEQ ID NO 1258
GNDGANIIY SEQ ID NO 383 GNSGNDVII SEQ ID NO: 821 GNDGNDVIV SEQ ID NO 1259
GDDGANIIY SEQ ID NO 384 GDSGNDVII SEQ ID NO: 822 GDDGNDVIV SEQ ID NO 1260
GGAGNDVIY SEQ ID NO 385 GGGGNDVII SEQ ID NO: 823 GGAGDDVIV SEQ ID NO 1261
GNAGNDVIY SEQ ID NO 386 GNGGNDVII SEQ ID NO: 824 GNAGDDVIV SEQ ID NO 1262
GDAGNDVIY SEQ ID NO 387 GDGGNDVII SEQ ID NO: 825 GDAGDDVIV SEQ ID NO 1263
GGSGNDVIY SEQ ID NO 388 GGDGNDVII SEQ ID NO: 826 GGSGDDVIV SEQ ID NO 1264
GNSGNDVIY SEQ ID NO 389 GNDGNDVII SEQ ID NO: 827 GNSGDDVIV SEQ ID NO 1265
GDSGNDVIY SEQ ID NO 390 GDDGNDVII SEQ ID NO: 828 GDSGDDVIV SEQ ID NO 1266
GGGGNDVIY SEQ ID NO 391 GGAGDDVII SEQ ID NO: 829 GGGGDDVIV SEQ ID NO 1267
GNGGNDVIY SEQ ID NO 392 GNAGDDVII SEQ ID NO: 830 GNGGDDVIV SEQ ID NO 1268
GDGGNDVIY SEQ ID NO 393 GDAGDDVII SEQ ID NO: 831 GDGGDDVIV SEQ ID NO 1269 Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO) Sequence (SI .Q I I) NO)
GGDGNDVIY SEQ ID NO 394 GGSGDDVII SEQ ID NO 832 GGDGDDVIV SEQ ID NO 1270
GNDGNDVIY SEQ ID NO 395 GNSGDDVII SEQ ID NO 833 GNDGDDVIV SEQ ID NO 1271
GDDGNDVIY SEQ ID NO 396 GDSGDDVII SEQ ID NO 834 GDDGDDVIV SEQ ID NO 1272
GGAGDDVIY SEQ ID NO 397 GGGGDDVII SEQ ID NO 835 GGAGADVIV SEQ ID NO 1273
GNAGDDVIY SEQ ID NO 398 GNGGDDVII SEQ ID NO 836 GNAGADVIV SEQ ID NO 1274
GDAGDDVIY SEQ ID NO 399 GDGGDDVII SEQ ID NO 837 GDAGADVIV SEQ ID NO 1275
GGSGDDVIY SEQ ID NO 400 GGDGDDVII SEQ ID NO 838 GGSGADVIV SEQ ID NO 1276
GNSGDDVIY SEQ ID NO 401 GNDGDDVII SEQ ID NO 839 GNSGADVIV SEQ ID NO 1277
GDSGDDVIY SEQ ID NO 402 GDDGDDVII SEQ ID NO 840 GDSGADVIV SEQ ID NO 1278
GGGGDDVIY SEQ ID NO 403 GGAGADVII SEQ ID NO 841 GGGGADVIV SEQ ID NO 1279
GNGGDDVIY SEQ ID NO 404 GNAGADVII SEQ ID NO 842 GNGGADVIV SEQ ID NO 1280
GDGGDDVIY SEQ ID NO 405 GDAGADVII SEQ ID NO 843 GDGGADVIV SEQ ID NO 1281
GGDGDDVIY SEQ ID NO 406 GGSGADVII SEQ ID NO 844 GGDGADVIV SEQ ID NO 1282
GNDGDDVIY SEQ ID NO 407 GNSGADVII SEQ ID NO 845 GNDGADVIV SEQ ID NO 1283
GDDGDDVIY SEQ ID NO 408 GDSGADVII SEQ ID NO 846 GDDGADVIV SEQ ID NO 1284
GGAGADVIY SEQ ID NO 409 GGGGADVII SEQ ID NO 847 GGAGNNVIV SEQ ID NO 1285
GNAGADVIY SEQ ID NO 410 GNGGADVII SEQ ID NO 848 GNAGNNVIV SEQ ID NO 1286
GDAGADVIY SEQ ID NO 411 GDGGADVII SEQ ID NO 849 GDAGNNVIV SEQ ID NO 1287
GGSGADVIY SEQ ID NO 412 GGDGADVII SEQ ID NO 850 GGSGNNVIV SEQ ID NO 1288
GNSGADVIY SEQ ID NO 413 GNDGADVII SEQ ID NO 851 GNSGNNVIV SEQ ID NO 1289
GDSGADVIY SEQ ID NO 414 GDDGADVII SEQ ID NO 852 GDSGNNVIV SEQ ID NO 1290
GGGGADVIY SEQ ID NO 415 GGAGNNVII SEQ ID NO 853 GGGGNNVIV SEQ ID NO 1291
GNGGADVIY SEQ ID NO 416 GNAGNNVII SEQ ID NO 854 GNGGNNVIV SEQ ID NO 1292
GDGGADVIY SEQ ID NO 417 GDAGNNVII SEQ ID NO 855 GDGGNNVIV SEQ ID NO 1293
GGDGADVIY SEQ ID NO 418 GGSGNNVII SEQ ID NO 856 GGDGNNVIV SEQ ID NO 1294
GNDGADVIY SEQ ID NO 419 GNSGNNVII SEQ ID NO 857 GNDGNNVIV SEQ ID NO 1295
GDDGADVIY SEQ ID NO 420 GDSGNNVII SEQ ID NO 858 GDDGNNVIV SEQ ID NO 1296
GGAG VIY SEQ ID NO 421 GGGGNNVII SEQ ID NO 859 GGAGDNVIV SEQ ID NO 1297
GNAG VIY SEQ ID NO 422 GNGGNNVII SEQ ID NO 860 GNAGDNVIV SEQ ID NO 1298
GDAG VIY SEQ ID NO 423 GDGGNNVII SEQ ID NO 861 GDAGDNVIV SEQ ID NO 1299
GGSG NVIY SEQ ID NO 424 GGDGNNVII SEQ ID NO 862 GGSGDNVIV SEQ ID NO 1300
GNSG NVIY SEQ ID NO 425 GNDGNNVII SEQ ID NO 863 GNSGDNVIV SEQ ID NO 1301
GDSG NVIY SEQ ID NO 426 GDDGNNVII SEQ ID NO 864 GDSGDNVIV SEQ ID NO 1302
GGGG VIY SEQ ID NO 427 GGAGDNVII SEQ ID NO 865 GGGGDNVIV SEQ ID NO 1303
GNGG VIY SEQ ID NO 428 GNAGDNVII SEQ ID NO 866 GNGGDNVIV SEQ ID NO 1304
GDGG VIY SEQ ID NO 429 GDAGDNVII SEQ ID NO 867 GDGGDNVIV SEQ ID NO 1305
GGDG VIY SEQ ID NO 430 GGSGDNVII SEQ ID NO 868 GGDGDNVIV SEQ ID NO 1306
GNDG VIY SEQ ID NO 431 GNSGDNVII SEQ ID NO 869 GNDGDNVIV SEQ ID NO 1307
GDDG VIY SEQ ID NO 432 GDSGDNVII SEQ ID NO 870 GDDGDNVIV SEQ ID NO 1308
GGAGD VIY SEQ ID NO 433 GGGGDNVII SEQ ID NO 871 GGAGANVIV SEQ ID NO 1309
GNAGD VIY SEQ ID NO 434 GNGGDNVII SEQ ID NO 872 GNAGANVIV SEQ ID NO 1310
GDAGD VIY SEQ ID NO 435 GDGGDNVII SEQ ID NO 873 GDAGANVIV SEQ ID NO 1311
GGSGDNVIY SEQ ID NO 436 GGDGDNVII SEQ ID NO 874 GGSGANVIV SEQ ID NO 1312
GNSGDNVIY SEQ ID NO 437 GNDGDNVII SEQ ID NO 875 GNSGANVIV SEQ ID NO 1313
GDSGDNVIY SEQ ID NO 438 GDDGDNVII SEQ ID NO 876 GDSGANVIV SEQ ID NO 1314
GGGGD VIY SEQ ID NO 439 GGAGANVII SEQ ID NO 877 GGGGANVIV SEQ ID NO 1315
GNGGD VIY SEQ ID NO 440 GNAGANVII SEQ ID NO 878 GNGGANVIV SEQ ID NO 1316
GDGGD VIY SEQ ID NO 441 GDAGANVII SEQ ID NO 879 GDGGANVIV SEQ ID NO 1317
GGDGD VIY SEQ ID NO 442 GGSGANVII SEQ ID NO 880 GGDGANVIV SEQ ID NO 1318
GNDGD VIY SEQ ID NO 443 GNSGANVII SEQ ID NO 881 GNDGANVIV SEQ ID NO 1319
GDDGD VIY SEQ ID NO 444 GDSGANVII SEQ ID NO 882 GDDGANVIV SEQ ID NO 1320
GGAGA VIY SEQ ID NO 445 GGGGANVII SEQ ID NO 883 GDEASDLFF SEQ ID NO 1321
GNAGANVIY SEQ ID NO 446 GNGGANVII SEQ ID NO 884 GDLASDLFF SEQ ID NO 1322 Sequence (SI .Q I I) NO) Sequence (SI .Q I D NO) Sequence (SI .Q I I) NO)
GDAGA VIY SEQ ID NO: 447 GDGGANVII SEQ ID NO: 885 GDNASDLFF SEQ ID NO 1323
GGSGANVIY SEQ ID NO: 448 GGDGANVII SEQ ID NO: 886 GDEASDLFT SEQ ID NO 1324
GNSGANVIY SEQ ID NO: 449 GNDGANVII SEQ ID NO: 887 GDLASDLFT SEQ ID NO 1325
GDSGANVIY SEQ ID NO: 450 GDDGANVII SEQ ID NO: 888 GDNASDLFT SEQ ID NO 1326
GGGGANVIY SEQ ID NO: 451 GGAGNDTLV SEQ ID NO: 889 GDEASDLFN SEQ ID NO 1327
GNGGA VIY SEQ ID NO: 452 GNAGNDTLV SEQ ID NO: 890 GDLASDLFN SEQ ID NO 1328
GDGGA VIY SEQ ID NO: 453 GDAGNDTLV SEQ ID NO: 891 GDNASDLFN SEQ ID NO 1329
GGDGANVIY SEQ ID NO: 454 GGSGNDTLV SEQ ID NO: 892 GDEASDLFD SEQ ID NO 1330
GNDGA VIY SEQ ID NO: 455 GNSGNDTLV SEQ ID NO: 893 GDLASDLFD SEQ ID NO 1331
GDDGA VIY SEQ ID NO: 456 GDSGNDTLV SEQ ID NO: 894 GDNASDLFD SEQ ID NO 1332
GGAGNDTLI SEQ ID NO: 457 GGGGNDTLV SEQ ID NO: 895 GDEASDLFK SEQ ID NO 1333
GNAGNDTLI SEQ ID NO: 458 GNGGNDTLV SEQ ID NO: 896 GDLASDLFK SEQ ID NO 1334
GDAGNDTLI SEQ ID NO: 459 GDGGNDTLV SEQ ID NO: 897 GDNASDLFK SEQ ID NO 1335
GGSGNDTLI SEQ ID NO: 460 GGDGNDTLV SEQ ID NO: 898 GDEASDLFS SEQ ID NO 1336
GNSGNDTLI SEQ ID NO: 461 GNDGNDTLV SEQ ID NO: 899 GDLASDLFS SEQ ID NO 1337
GDSGNDTLI SEQ ID NO: 462 GDDGNDTLV SEQ ID NO: 900
[0043] As used herein, the term "precipatable-beta roll cassette" (PBRC) refers to an amino acid sequence comprising at least one PBRT. In certain embodiments, a PBRC will comprise at least two PBRTs. In certain embodiments, a PBRC will comprise at least 3 PBRTs, at least 4 PBRTs, at least 5 PBRTs, at least 6 PBRTs, at least 7 PBRTs, at least 8 PBRTs, at least 9 PBRTs, at least 10 PBRTs, at least 11 PBRTs, at least 12 PBRTs, at least 13 PBRTs, at least 14 PBRTs, at least 15 PBRTs, at least 16 PBRTs, at least 17 PBRTs, at least 18 PBRTs, at least 19 PBRTs, at least 20 PBRTs, or 20 or more PBRTs. In certain embodiments, the
PBRCs described herein will comprise a plurality of precipatable beta roll tags arranged in a tandem repeat. For example, in certain embodiments, the PBRCs described herein can
comprise at least 2 PBRTs, at least 3 PBRTs, at least 4 PBRTs, at least 5 PBRTs, at least 6
PBRTs, at least 7 PBRTs, at least 8 PBRTs, at least 9 PBRTs, at least 10 PBRTs, at least 11 PBRTs, at least 12 PBRTs, at least 13 PBRTs, at least 14 PBRTs, at least 15 PBRTs, at least 16 PBRTs, at least 17 PBRTs, at least 18 PBRTs, at least 19 PBRTs, at least 20 PBRTs, or 20 or more PBRTs in tandem repeat. In certain embodiments, a PBRC can comprise at least two PBRCs separated by a linking amino acid sequence. Where a linking amino acid sequence in present between two PBRTs, a PBRT located at either end of the linking sequence can be an individual PBRT or it can be a PBRT that is part of a tandem arrangement of two or more
PBRTs.
[0044] The PBRCs can comprise polymeric or oligomeric repeats of a PBRT. In certain
embodiments, the PBRCs described herein can comprise one or more different PBRTs. In one embodiment, all of the PBRTs comprised in a PBRC are identical in amino acid sequence. In one embodiment, all of the PBRTs comprised in a PBRC have different amino acid sequences. In one embodiment, at least one PBRT comprised in a PBRC has a different amino acid sequence as compared to another PBRT in the PBRC. Thus, in certain
embodiments, the PBRCs described herein can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 different PBRTs.
[0045] In certain embodiments, a PBRC can also comprise a capping sequence ("CS") refers to the an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 3. The term "capping sequence" also refers to a capping sequence having the amino acid sequence of any of SEQ ID NO: 4-23.
[0046] Without wishing to be bound to theory, in some embodiments of the invention, the ability of polypeptide comprising one or more PBRT to undergo reversible Ca2+
precipitation, can require that the one or more PBRTs be located N-terminally or C- terminally to a capping sequence. Thus, in certain embodiments, the capping sequence is an amino acid sequence, which, when located C-terminally or N- terminally to one or more PBRTs, allows the one or more PBRTs bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the PBRC.
[0047] Thus in certain embodiments, a PBRC will comprise at least 2 PBRTs, at least 3 PBRTs, at least 4 PBRTs, at least 5 PBRTs, at least 6 PBRTs, at least 7 PBRTs, at least 8 PBRTs, at least 9 PBRTs, at least 10 PBRTs, at least 11 PBRTs, at least 12 PBRTs, at least 13 PBRTs, at least 14 PBRTs, at least 15 PBRTs, at least 16 PBRTs, at least 17 PBRTs, at least 18 PBRTs, at least 19 PBRTs, at least 20 PBRTs, or 20 or more PBRTs, all of which are located N-terminally to a CS. In certain embodiments, a PBRC will comprise 2, at least 3 PBRTs, at least 4 PBRTs, at least 5 PBRTs, at least 6 PBRTs, at least 7 PBRTs, at least 8 PBRTs, at least 9 PBRTs, at least 10 PBRTs, at least 11 PBRTs, at least 12 PBRTs, at least 13 PBRTs, at least 14 PBRTs, at least 15 PBRTs, at least 16 PBRTs, at least 17 PBRTs, at least 18 PBRTs, at least 19 PBRTs, at least 20 PBRTs, or 20 or more PBRTs, all of which are located C-terminally to a CS. [0048] In one embodiment, a PBRC is an amino acid sequence comprising at least five tandem PBRTs situated N-terminally to a capping sequence.
[0049] In one embodiment, a PBRC is an amino acid sequence comprising at least six to about 16 tandem PBRTs situated N-terminally to a capping sequence.
[0050] In one embodiment, a precipatable beta roll cassette is an amino acid sequence comprising 17 or more tandem PBRTs situated N-terminally to a capping sequence.
[0051] In one embodiment, the capping sequence comprises the sequence
INAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQAGI EKLVEAMAQYPD (SEQ ID NO: 3) which is a C-terminal sequence on the block V beta roll domain of the adenylate cyclase toxin of B. pertussis.
[0052] In another embodiment, a PBRC comprises the amino acid sequence
GGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYINAGADQLW
FARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQAGIEKLVEAMAQ
YPD (SEQ ID NO: 4). In another embodiment, a PBRC comprises the amino acid sequence
GGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTL
YGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDT
LYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYINAGADQ
LWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQAGIEKLVEAM
AQYPD (SEQ ID NO: 5). In another embodiment, the capping sequence comprises the sequence
INAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEAIHAANQAIDPAGI EKLVEAMAQYPD (SEQ ID NO: 6) (adenylate cyclase-hemolysin [Bordetella
bronchiseptica]). In another embodiment, the capping sequence comprises the sequence INAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEAIHAANQAIDPAGI EKLVEAMAQYPD (SEQ ID NO: 7) (adenylate cyclase-hemolysin [Bordetella
bronchiseptica]). In another embodiment, the capping sequence comprises the sequence INAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEAIHAANQTVDPAGI EKLVEAMAQYPD (SEQ ID NO: 8) (adenylate cyclase hemolysin [Bordetella
bronchiseptica]). In another embodiment, the capping sequence comprises the sequence QLWFSKSGSDLEVRVVGTDDAVTVAGWYSGAEHHMDSIETADGTVLLDSMVDRLV QAMAGF (SEQ ID NO: 9) (Azospirillum sp. B510 calcium binding hemolysin protein). In another embodiment, the capping sequence comprises the sequence ADQLWFRHVGNDLEISILGTGDTATVRDWYLGSRYQIEQIRVDDGRTLVNADVEKL VQAMA (SEQ ID NO: 10) (hemolysin-type calcium-binding region Burkholderia cenocepacia MCO-3). In another embodiment, the capping sequence comprises the sequence ADQLWFRHVGNDLEISILGSSDTATVRDWYSGSRYQIEQIRLDDGRTLVNADVEKLV QAM A (SEQ ID NO: 11) (hemolysin-type calcium-binding region [Burkholderia ambifaria MC40-6]). In another embodiment, the capping sequence comprises the sequence
DARQTNLWFSQVGKDLQIDVLGSTDQVTVKDWYAGADNRVERIKTADGKTLYDSD VDKLVQAMASF (SEQ ID NO: 12) (calcium binding secreted hemolysin
protein) [Herbaspirillum seropedicae SmRl]). In another embodiment, the capping sequence comprises the sequence
DARQTNLWFSQVGKDLQIDVLGSTDQVTVKDWYAGADNRVERIKTADGKTLYDSD VDKLVQAMASF (SEQ ID NO: 13) (calcium binding secreted hemolysin protein
[Herbaspirillum seropedicae SmRl]). In another embodiment, the capping sequence comprises the sequence
ELWFSRENNDLIIKSLLSEDKVTVQNWYSHQDHKIENIRLSNEQTLVSTQVEKMVES MAGF (SEQ ID NO: 14) (RTX toxin protein [Actinobacillus pleuropneumoniae serovar). In another embodiment, the capping sequence comprises the sequence
EELWFSRDGNDLQINVIGTDNQVEISDWYSGVNYQLDKVQVGDSVLLNTQLEQLVS AMASF (SEQ ID NO: 15) (hemolysin-type calcium binding protein [Shewanella
piezotolerans WP3]). In another embodiment, the capping sequence comprises the sequence GLSELWFSRENNDLIIKSLLSEDKVTVQNWYSHQDHKIENIRLSNEQMLVSTQVEKM VESMAGF (SEQ ID NO: 16) (RTX toxin protein [Actinobacillus pleuropneumoniae serovar 10). In another embodiment, the capping sequence comprises the sequence
EDLWFSRDGNNLQINIIGTDDQVEVNNWYNDTNYQLDQIQVGGSVLLNNQLEQLVS AMASF (SEQ ID NO: 17) (RTX toxin [Shewanella violacea DSS12]). In another embodiment, the capping sequence comprises the sequence
ELWFSRENNDLIIKSLLSEDKVTVQNWYSHQDHKIENIRLSNEQTLVSTQVEKMVES MASF (SEQ ID NO: 18) (RTX toxin protein [Actinobacillus pleuropneumoniae serovar 6). In another embodiment, the capping sequence comprises the sequence
ADNFWFVKSGNDLEIDILGTHQQVTVADWFLGGSYQLQEIKAGGLELDTQVTQLVQ AMATY (SEQ ID NO: 19) (protein BRAD06535 [Bradyrhizobium sp. ORS278]). In another embodiment, the capping sequence comprises the sequence
ELWFSRENNDLIIKSLLSEDKVTVQNWYSHQDHKIENIRLSNEQTLVSTQVEKMVES MAGF (SEQ ID NO: 20) ([Actinobacillus pleuropneumoniae L20]). In another embodiment, the capping sequence comprises the sequence
LWFSRENNDLIIKSLLSEDKVTVQNWYSHQDHKIENIRLSNEQTLVSTQVEKMVESM AGF (SEQ ID NO: 21) (ApxIVA [Actinobacillus pleuropneumoniae]). In another embodiment, the capping sequence comprises the sequence
LWFRKSGNNLEVSIIGTSDKLVMSNWYAGSQYQVERFQAGDGKALQANQVQSLVQ AM ASF (SEQ ID NO: 22) (hemolysin-type calcium-binding protein [Xanthomonas axonopodis pv. citri str. 306]). In another embodiment, the capping sequence comprises the sequence
ELWFSRENNDLIIKSLLSEDKVTVQNWYSHQDHKIENIRLSNEQTLVSTQVEKMVES MAGF (SEQ ID NO: 23) (RTX toxin IVA [Actinobacillus pleuropneumoniae])
[0053] Without wishing to be bound to theory, in some embodiments of the invention, the ability of polypeptide comprising one or more PBRT to undergo reversible Ca2+
precipitation, can require that the one or more PBRTs be located N-terminally or C- terminally to a stabilizing polypeptide. It is known that when a certain stabilizing
polypeptides (e.g. GFP, maltose binding protein) are attached to the C-terminus of the beta roll, calcium- induced folding can occur (Blenner et al., Journal of Molecular Biology. 400 (2010), pp 244-256; Szilvay et al, Biochemistry, 48 (2009), pp 11273-11282).
[0054] Thus, in one embodiment, a PBRC is an amino acid sequence comprising one or more PBRTs located N-terminally or C-terminally to a stabilizing polypeptide, wherein the stabilizing polypeptide cane be, but is not limited to, glutathione S-transferase (GST), maltose E binding protein (MBP), Green Fluorescent Protein (GFP), and variants thereof. In still a further embodiment, the stabilizing polypeptide is an amino acid sequence of any amino acid composition wherein the sequence comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, or at least 68 amino acids. [0055] In another aspect of the invention, the PBRCs described herein can further comprise one or more cleavage sites. In certain embodiments, the cleavage site can be positioned C- terminally or N-terminally to a PBRC so as to allow for cleavage of a PBRC from a linked purification moiety (e.g. a polypeptide purification moiety linked to a PBRC as part of a fusion protein). In embodiments where the PBRC comprises more than one cleavage site, a first cleavage site can be positioned C-terminally or N-terminally to a PBRC so as to allow for cleavage of a PBRC from a linked purification moiety (e.g. a polypeptide purification moiety linked to a PBRC as part of a fusion protein) and a second cleavage site can be positioned between a PBRT and a capping sequence or a PBRT and a stabilizing polypeptide so as to allow so as to allow for cleavage of the capping sequence or the stabilizing polypeptide from the one or more PBRTs in the PBRC. In certain embodiments, cleavage at such cleavage sites can be useful for purification of a purification moiety of interest.
[0056] In one embodiment, the cleavage site is a proteolytic cleavage site. Exemplary proteolytic cleavage sites, include, but are not limited to Factor Xa, thrombin, or
enterokinase. In another embodiment, the cleavage site is a signal peptidase cleavage site. In another embodiment, the cleavage site is a self cleaving intein cleavage site (Amitai et al., Proceedings of the National Academy of Sciences, vol. 106, no. 27, pp. 11005 -11010, Jul. 2009; Hiraga et al., Journal of Molecular Biology, vol. 393, no. 5, pp. 1106-1117, Nov. 2009). Any other specific cleavage sites known in the art can be used in connection with the methods described herein.
[0057] The PBRTs or PBRCs described herein may be linked to a purification moiety by any means known in the art. The PBRTs or PBRCs described herein ca be located at any site in a polypeptide comprising a purification moiety of interest, including a location that is N- terminal, a location that is C-terminal or a location within the sequence of the purification moiety of interest.
[0058] In addition to fusion proteins comprising the PBRTs or PBRCs described herein, the PBRTs or PBRCs described herein can also be chemically linked to purification moieties other than by means of a fusion protein. Thus, reference to a PBRC linked purification moiety, or to a PBRT linked purification moiety encompasses for purification moieties linked to a PBRC or PBRT by peptide linkage (e.g. as a fusion protein) or by non-peptide bond chemical linkage. [0059] The chemical modification of PBRTs or PBRCs described herein can be performed according to any method known in the art.. For example, amides of the PBRTs or PBRCs described herein can be prepared by techniques well known in the art for converting a carboxylic acid group or precursor, to an amide. One method for amide formation at the C- terminal carboxyl group is to cleave the polypeptide, or fusion thereof from a solid support with an appropriate amine, or to cleave in the presence of an alcohol, yielding an ester, followed by aminolysis with the desired amine.
[0060] Salts of carboxyl groups of the PBRTs or PBRCs described herein can be prepared by contacting the polypeptide, or fusion thereof with one or more equivalents of a desired base such as, for example, a metallic hydroxide base, e.g., sodium hydroxide; a metal carbonate or bicarbonate base such as, for example, sodium carbonate or sodium bicarbonate; or an amine base such as, for example, triethylamine, triethanolamine, and the like.
[0061] N-acyl derivatives of an amino group of the PBRTs or PBRCs described herein can be prepared by utilizing an N-acyl protected amino acid for the final condensation, or by acylating a protected or unprotected polypeptide, or fusion thereof. O-acyl derivatives can be prepared, for example, by acylation of a free hydroxy polypeptide or polypeptide resin. Either acylation can be carried out using standard acylating reagents such as acyl halides, anhydrides, acyl imidazoles, and the like. Both N- and O-acylation can be carried out together, if desired.
[0062] Formyl-methionine, pyroglutamine and trimethyl-alanine can be substituted at the N- terminal residue of PBRTs or PBRCs described herein. Other amino-terminal modifications include aminooxypentane modifications.
[0063] Such chemical linkages can be useful for purifying non-peptide molecules such as lipids, oligonucleotides and carbohydrates, small organic or inorganic molecules, proteins, single-stranded or double-stranded oligonucleotides, polynucleotides, metals (e.g. cobalt, zinc, nickel or copper) and the like. The chemically modified PBRTs or PBRCs described herein can be assayed for the ability to bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the PBRT or PBRC using methods known to those skilled in the art.
[0064] The PBRTs and PBRCs described herein can also be coupled with a radioisotope or enzymatic label to facilitate their detection. For example, the PBRTs or PBRCs described herein can be isotopically-labeled where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring). Suitable radionuclides that may be incorporated in compounds of the present invention include but are not limited to 2H (also written as D for deuterium), 3H (also written as T for tritium), nC, 13C, 14C, 13N, 15N, 150, 170, 180, 18F, 35S, 36C1, 82Br, 75Br, 76Br, 77Br, 1231, 124I, 125I and 131I. The radionuclide that is incorporated in the instant radio-labeled compounds can depend on the specific application of that radio-labeled compound.
[0065] Alternatively, the PBRTs or PBRCs described herein can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In another embodiment, the PBRTs or PBRCs described herein can be labeled with a fluorescent dye, spin label, heavy metal or radio-labeled peptides.
[0066] Esters of carboxyl groups of the PBRTs or PBRCs described herein can also be prepared by any of the usual methods known in the art.
[0067] The methods and compositions described herein are useful in a broad range of bioseparation applications. The methods and compositions described herein can be used for rapid expression and purification of a purification moiety linked to a PBRC. Such PBRC linked purification moieties can be expressed in any number of expression systems, including in vitro and in vivo expression systems. Exemplary in vivo expression systems suitable for expressing the PBRC linked purification moieties described herein, include, but are not limited to, bacterial systems, yeast systems, and mammalian systems.
[0068] In one aspect, the invention relates to a method for purifying purification moieties (e.g. a PBRC or a purification moiety linked to a PBRC). In one aspect, the methods described herein can be used for purifying one or more purification moieties from a heterogeneous mixture of biomaterials in a sample. In another aspect, the methods descried herein can be used to purify chemically synthesized purification moieties or in-vitro synthesized purification moieties.
[0069] In certain embodiments, the bioseparation methods described herein can comprise expressing a PBRT linked purification moiety (e.g. a PBRC fusion protein) in a cellular expression system (e.g. a bacterial cell). The PBRC linked purification moiety can then be released into a medium by cell lysis. Any method of cell lysis known in the art can be used in conjunction with the methods described herein, including, but not limited to chemical lysis (e.g. detergents) or physical methods (e.g. sonication or French press). In certain embodiments, the PBRC or PBRC linked purification moiety can be expressed in an in-vitro expression system (e.g. a rabbit reticulocyte system) such that the purification moiety is expressed into the expression system medium.
[0070] After expression of the PBRC linked purification moiety, bioseparation can be achieved by increasing the free Ca2+ concentration in the medium comprising the PBRC linked purification moiety to induce precipitation of the PBRC linked purification moiety, followed by removing material that does not precipitate from the medium and then resuspending the precipitated material in a medium having a reduced Ca2+ concentration or in a medium having a reduced concentration of free Ca2+ (e.g. a medium comprising a Ca2+ chelators such as EDTA). These steps can be repeated until the desired level of purity is reached.
[0071] In another embodiment, the PBRC linked purification moiety can be expressed in a cellular expression system, and bioseparation can be achieved by increasing the free Ca2+ concentration within the cell prior to cellular lysis. Many methods for increasing intracellular Ca2+ concentrations are known in the art, including, but not limited to adding Ca2+ to a cellular medium, with or without presence of ionophores or cell permeabilization agents. In such embodiments, the cells can then be subjected to lysis conditions (e.g. chemical lysis or physical lysis) and the resulting precipitate can be recovered. Precipitated PBRC or the PBRC linked purification moieties can then be recovered by reducing the free Ca2+ concentration (e.g. through the addition of a Ca2+ chelator) to induce solubilization of the purification moieties from the precipitate and bioseparation can be achieved by eliminating the precipitate. Precipitation and solubilization steps can be repeated until a desired level of purity is reached.
[0072] In still other embodiments, the PBRC linked purification moieties described herein can further comprise a peptide sequence to induce secretion into the periplasm of a cell (e.g. an E. coli cell) or to the medium outside of a cell. Where the PBRC linked purification moiety is secreted to the periplasm of a cell, cell lysis may be required for further purification of the PBRC linked purification moiety. Where the PBRC linked purification moiety is secreted into the medium outside of the cell, purification can be achieved without cell lysis by eliminating intact cells (e.g. by centrifugation) and purification of the PBRC linked purification moiety from the extracellular medium by increasing the free Ca2+ concentration of the supernatant. [0073] The adjustment of conditions during the purification process can be achieved by numerous methods, including, but not limited to, adjusting the temperature, pH or salt concentration of the aqueous media.
[0074] The methods described herein can be used to purify purification moieties of any size. A purified PBRT linked purification moiety can contain less than about 50%, less than about 75%, or less than about 90%, of the materials with which it was originally associated.
[0075] In one embodiment, a purification moiety of interest can be linked to PBRC comprising a cleavable peptide sequence (e.g. a self-cleaving peptide sequence such an intein) positioned between the PBRC and the purification moiety. Once the PBRC linked purification moiety is expressed in an expression system, the PBRC linked purification moiety can be recovered using standard techniques as either a homogenous mixture or as a heterogeneous sample. The mixture can then be exposed to calcium to induce precipitation of the PBRC linked purification moiety. The PBRC linked purification moiety can then be resuspended in buffer that has reduced Ca2+, that has reduced free Ca2+ or that contains a calcium chelator (e.g. EDTA). The PBRC linked purification moiety can then ne subjected condition that cause cleavage to separate the PBRC from the purification moiety and calcium can be once again added to the mixture. This will precipitate out the PBRC moiety and thereby leaving behind a sample of purified purification moiety of interest.
[0076] In another embodiment, PBRC linked purification moiety can be a moiety which binds a second molecule and the second molecule can be used to remove the purification moiety from the sample (e.g. a resin or beads coated with the second molecule) after induced cleavage at a site between the purification moiety and the PBRC.
[0077] In some embodiments, immobilization of the PBRC linked purification moieties described herein or its binding proteins can be used to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Immobilization of the PBRC linked purification moieties described herein can be by linking to a solid support, including a plastic or glass plate or bead, a chromatographic resin, a filter or a membrane. Methods of attachment of proteins, or membranes containing same, to such supports are well known in the art. Immobilization of the PBRC linked purification moieties described herein can also be accomplished in any vessel suitable for containing the reactants. Examples include microtiter plates, test tubes, and micro-centrifuge tubes. [0078] In one embodiment, a fusion protein can be provided which adds a domain that allows the PBRT linked purification moiety described herein to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads or glutathione derivatized microtiter plates, which are then combined with the cell lysates, and the mixture incubated under conditions conducive to complex formation.. Following incubation, the beads can be washed to remove any unbound fraction,. Alternatively, the complexes can be dissociated from the matrix using standard electrophoretic techniques.
[0079] The methods described herein depend, in part on the finding that PBRCs undergo a reversible Ca2+ binding dependent transition. PBRCs or PBRC linked purification moieties undergo reversible precipitation at a Ca2+ concentration (or free Ca2+) phase transition concentration.
[0080] The transition concentrations reversible and the isolated precipatable beta-roll tags or purification moieties comprising a precipatable beta-roll tag can be completely resolubilized in a medium below a certain Ca2+ concentration (or free Ca2+) transition concentration, through, for example the addition of a calcium chelator into the medium comprising the PBRC or PBRC linked purification moiety.
[0081] The concentration of Ca2+ required to induce reversible precipitation of a PBRC or PBRC linked purification moiety can be readily determined by adding increasing amounts of Ca2+ until such time as the PBRC or PBRC linked purification moiety begins to precipitate from a the medium. One can readily determine the extent of precipitation by centrifuging the medium. In one embodiment, the amount of Ca2+ required to induce reversible precipitation a PBRC of PBRC linked purification moiety will be about lmM Ca2+, more than about ImM Ca2+, more than about 5mM Ca2+, more than about lOmM Ca2+, more than about 20mM Ca2+, more than about 30mM Ca2+, more than about 50mM Ca2+, more than about 75mM Ca2+, more than about 100 mM Ca2+, more than about 150mM Ca2+, more than about 200mM Ca2+, or more than about 500mM Ca2+. In certain embodiments, the amount of Ca2+ required to induce precipitation of a PBRC or a PBRC linked purification moiety can increase as a function of the number of PBRTs in the PBRC. For example, a PBRC linked purification moiety comprising 8 PBRTs may precipitate in 150mM Ca2+ wherein a PBRC linked purification moiety comprising 17 PBRTs may precipitate in 25mM Ca2+. One of skill in the art will readily be capable of determining the amount of Ca2+ required to precipitate a particular PBRC or a particular PBRC linked purification moiety simply by titrating increasing concentrations of Ca2+. [0082] The concentration of Ca2+ required to reverse precipitation of a PBRT or PBRC or of a PBRT or PBRC linked purification moiety can be readily determined by reducing the concentration of free Ca2+ in a medium until such time as a precipitated PBRC or PBRC linked purification moiety begins to solubilize into the medium. One can readily determine the extent of precipitation by centrifuging the medium. In one embodiment, the amount of free Ca2+ in the medium required to solubilize a precipitated PBRT or PBRC or of a PBRT or PBRC linked purification moiety will be less than about ImM Ca2+, less than about ImM Ca2+, less than about 5mM Ca2+, less than about lOmM Ca2+, less than about 20mM Ca2+, less than about 30mM Ca2+, less than about 50mM Ca2+, less than about 75mM Ca2+, less than about 100 mM Ca2+, less than about 150mM Ca2+, less than about 200mM Ca2+, or less than about 500mM Ca2+. In certain embodiments, the free Ca2+ concentration required to reverse precipitation of a PBRC or a PBRC linked purification moiety can correlated to the number of PBRTs in the PBRC. For example, a PBRC linked purification moiety comprising 8 PBRTs may become soluble in a higher free Ca2+ concentration as compared to a PBRC linked purification moiety comprising 17 PBRTs. One of skill in the art will readily be capable of determining the Ca2+ concentration required to reverse precipitation a particular PBRC or a particular PBRC linked purification moiety simply by decreasing free Ca2+ concentrations.
[0083] The free Ca2+ concentration of a medium comprising a PBRC or a PBRC linked purification moiety can be reduced by adding one or more calcium chelators into the medium. Any number of calcium chelators can be used in the connection with the methods described herein. Examples of suitable calcium chelators include, but are not limited to EDTA, EGTA, and BAPTA. In one embodiment, the amount of a calcium chelator required to solubilize a precipitated PBRT or PBRC or of a PBRT or PBRC linked purification moiety will be about ImM Ca2+, more than about ImM Ca2+, more than about 5mM Ca2+, more than about lOmM Ca2+, more than about 20mM Ca2+, more than about 30mM Ca2+, more than about 50mM Ca2+, more than about 75mM Ca2+, more than about 100 mM Ca2+, more than about 150mM Ca2+, more than about 200mM Ca2+, or more than about 500mM Ca2+.
[0084] In addition to temperature and ionic strength, other environmental variables useful for modulating the solubility of PBRT or PBRC or of the PBRT linked purification moieties described herein include pH, the addition of organic solutes and solvents, side-chain ionization or chemical modification, and pressure. [0085] The PBRC linked purification moieties described herein can be further purified or isolated according to any method of protein purification or isolation known in the art. For example, PBRCs or PBRC linked purification moieties can be purified by various methods including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, precipitation and salting-out chromatography, extraction, and countercurrent distribution. For some purposes, the PBRCs or the PBRC linked purification moieties can be produced in a recombinant system in which the protein contains an additional sequence tag that facilitates purification, such as, but not limited to, a polyhistidine sequence, or a sequence that specifically binds to an antibody, such as FLAG and GST. In one embodiment, the PBRCs or the PBRC linked purification moieties can be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix. Alternatively, antibodies produced against the PBRTs or PBRCs, or a PBRC linked purification moiety or against polypeptides derived therefrom can be used as purification reagents.
[0086] The methods and compositions described herein can be useful for the detection of a broad range of purification moieties in biosensing applications. For example, the methods and compositions described herein can be used for the separation of protein of interest from a sample for detection of bimolecular interactions. In one embodiment, if a PBRC is linked to an antibody, the PBRC linked antibody can be added to a sample. Ca2+ can then be added to the sample to induce precipitation of the antibody such that antigen that interact with, or form a complex with, the antibody also precipitate upon the addition of Ca2+. The precipitate can then be collected and resuspended and the sample can be characterized. The presence and quantity of the target in the original sample, as well as any associated purification moieties, can be characterized and determined. Any antibodies, antibody fragments, antibody configurations, classes, or subclasses known in the art can be used in connection with the methods described herein. In another embodiment, if a PBRC is fused to a polypeptide capable of binding to a second purification moiety, it can be added to a sample. Ca2+ can then be added to the sample to induce precipitation of the polypeptide such that other purification moieties that interact with, or form a complex with, the polypeptide also precipitate upon the addition of Ca2+. The precipitate can then be collected and resuspended and the sample can be characterized. The presence and quantity of the target in the original sample, as well as any associated additional purification moieties, can be characterized and determined. [0087] The PBRC linked purification moieties described herein can be produced in prokaryotic or eukaryotic host cells by expression of nucleic acids encoding a polypeptide of this invention. The production of these polypeptides can also be done as part of a larger polypeptide.
[0088] The PBRC linked purification moieties described herein can also be synthesized in vitro, e.g., by the solid phase polypeptide synthetic method or by recombinant DNA approaches described herein. The solid phase polypeptide synthetic method is an established and widely used method. These PBRC or PBRC linked purification moieties described herein can be further purified by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on an anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; or ligand affinity chromatography.
[0089] The PBRC linked purification moieties described herein can also be produced using any in-vitro expression system known in the art or can be synthesized by chemical methods. Methods for expression of heterologous proteins in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in the art and are described further in Sambrook J et al.; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif; Gutte B and Merrifield R B, J. Am. Chem. Soc. 91 :501-02 (1969); Chaiken I M, CRC Crit. Rev. Biochem. 11 :255-301 (1981); Kaiser E T et al, Science 243: 187-92 (1989); Merrifield B, Science 232:341-47 (1986); Kent S B H, Ann. Rev. Biochem. 57:957-89 (1988); Offord, R. E. (1980) Semisynthetic Proteins, Wiley Publishing. Exemplary peptide synthesis methods known in the art include, but are not limited to those described in Stewart et al., Solid Phase Peptide Synthesis, Pierce Biotechnology, Inc., Rockford, 111., 1984; Bodanszky, Principles of Peptide Synthesis, Springer-Verlag, New York, 1984; and Pennington et al., Peptide Synthesis Protocols, Humana Press, Totowa, N.J., 1994). Additionally, many companies offer custom peptide synthesis services.
[0090] The PBRC linked purification moieties described herein can also be produced by direct chemical synthesis. For example, the PBRC linked purification moieties described herein can be produced as modified polypeptides, with nonpeptide moieties attached by covalent linkage to the N-terminus and/or C-terminus. In certain embodiments, either the carboxy-terminus or the amino-terminus, or both, are chemically modified. Common modifications of the terminal amino and carboxyl groups, include, but are not limited to acetylation and amidation, respectively. Amino-terminal modifications such as acylation (e.g., acetylation) or alkylation (e.g., methylation) and carboxy-terminal-modifications such as amidation, as well as other terminal modifications, including cyclization, can be incorporated into various embodiments. Certain amino-terminal and/or carboxy-terminal modifications and/or polypeptide extensions to the core sequence can provide advantageous physical, chemical, biochemical, and pharmacological properties, such as: enhanced stability, increased potency and/or efficacy, resistance to serum proteases, desirable pharmacokinetic properties, and others.
[0091] The PBRC linked purification moieties can be prepared using recombinant DNA and molecular cloning techniques. Genes encoding the PBRC linked purification moieties may be produced in heterologous host cells, particularly in the cells of microbial hosts. Any techniques for transfecting host cells and purifying proteins and polypeptides known in the art can be used in connection with the methods described herein. Exemplary epitope tags suitable for use with the methods described herein include, but are not limited to FLAG, HA, Myc and T7 epitope tags. The PBRTs, PBRCs or PBRC linked purification moieties described herein can be synthesized chemically using standard polypeptide synthesis techniques.
[0092] The invention also extends to the DNA expression vector comprising DNA coding for the PBRTs or PBRCs described herein, whether or not the encoded products further comprise a linked purification moiety. The invention also provides the expression vector comprising sequences coding for a PBRT or a PBRC configured to allow insertion of a DNA sequence downstream of the sequence coding for the PBRT or the PBRC so as to facilitate production of a fusion protein comprising a PBRT or a PBRC. For example, such vectors can comprise one or more cloning sites between the sequence coding for the PBRT or the PBRC to enable generation of an in-frame translation product. Such vectors may comprise multiple cloning sites in any of three reading frames. Methods for generating such expression vectors are well known in the art.
[0093] A variety of expression systems can be used to produce the PBRCs and PBRC linked purification moieties described herein. Such expression systems include vector based expression systems. Exemplary vector base expression systems suitable for use with the methods described herein include, but are not limited to, chromosomal, episomal and virus- derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from insertion elements, from yeast episomes, from viruses such as baculoviruses, retroviruses and vectors derived from combinations thereof such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
[0094] The expression system vectors may contain regulatory regions that regulate as well as engender expression. In general, any system or vector suitable to maintain, propagate or express polynucleotide or polypeptide in a host cell may be used for expression in this regard. Expression systems and expression vectors can contain regulatory sequences that direct high level expression of foreign proteins relative to the growth of the host cell. Regulatory sequences are well known to those skilled in the art and examples include, but are not limited to, those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of regulatory elements in the vector, for example, enhancer sequences. Any of these could be used to construct chimeric genes for production of the any of the binding peptides of the present invention. These chimeric genes could then be introduced into appropriate microorganisms via transformation to provide high level expression of the peptides.
[0095] A number of recombinant expression vectors can be used for expression of the PBRCs and PBRC linked purification moieties described herein. For example, the PBRT linked purification moieties described herein can be expressed in bacterial cells such as E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells, amphibian cells, or mammalian cells. Suitable host cells are well known to one skilled in the art. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, using, for example T7 promoter regulatory sequences and T7 polymerase.
[0096] Examples of E. coli expression vectors include pTrc (Amann E et al., Gene 69:301-15 (1988)) and pET l id (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) pp. 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 1 Id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
[0097] One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman S, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) pp. 119-28). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada K et al., Nucleic Acids Res. 20(Suppl.):2111-18 (1992)). Such alteration of nucleic acid sequences can be carried out by standard DNA synthesis techniques.
[0098] In another approach, a nucleic acid can be expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed B, Nature 329:840-41 (1987)) and pMT2PC (Kaufman R J et al, EMBO J. 6: 187-95 (1987)). When used in mammalian cells, the expression vector's control functions can be provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells, see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
[0099] A number of these methodologies can also be applied in vivo, systemically or locally, in a complex biological system such as a human. For example, increased copy number of nucleic acids PBRC or PBRC linked purification moieties described herein in expressible from (by DNA transfection), can be employed.
[00100] Nucleic acid purification moieties encoding PBRT or PBRC linked purification moieties described herein can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see for example, Gonzalez et al., Bioconjugate Chem. 10: 1068- 1074, 1999; Wang et al, International PCT Publication Nos. WO 03/47518 and WO
03/46185), poly(lactic-co-glycolic)ac-id (PLGA) and PLCA microspheres (see for example, U.S. Pat. No. 6,447,796 and U.S. Patent Application Publication No. US 2002130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722).
[00101] This invention may also be of use in the pharmaceutical/biotechnology industry where therapeutic compounds need to be purified in large quantities. This approach provides very pure product in a very quick manner. [00102] Purification moieties that can be linked to the PBRTs or PBRCs described herein can be any purification moiety, including a biologically active protein (e.g., a therapeutic peptide, protein or an enzyme useful in industrial biocatalysis).
[00103] The purification moieties suitable for use with the methods described herein can be of widely varying types, including, for example, peptides, non-peptide proteins, lipids, oligonucleotides and carbohydrates, or alternatively a ligand-binding protein or an active fragment thereof having binding affinity to a molecule selected from the group consisting of small organic or inorganic molecules, proteins, peptides, single-stranded or double-stranded oligonucleotides, polynucleotides, lipids, and carbohydrates.
[00104] Suitable purification moieties include, but are not limited to, molecules useful in medicine, agriculture and other scientific and industrial fields. For example, suitable molecules include those of interest in medicine, agriculture or other scientific or industrial fields. Examples of suitable proteins include enzymes utilized in replacement therapy;
hormones for promoting growth in animals, or cell growth in cell culture; and active proteinaceous substances used in various applications, e.g., in biotechnology or in medical diagnostics. One of skill in the art will recognize that many types of recombinant
polypeptides can be produced using the methods described herein. The present invention is not limited to any specific types of recombinant polypeptide described herein. Instead, it encompasses any and all recombinant polypeptides.
[00105] The PBRTs or PBRCs described herein can be joined to a purification moiety from any source or origin and can include a polypeptide found in prokaryotes, viruses, and eukaryotes, including fungi, plants, yeasts, insects, and animals, including mammals (e.g. humans). Purification moieties suitable for use with the methods described herein include, but are not limited to any polypeptide sequences, known or hypothetical or unknown, which can be identified using common sequence repositories. Examples of such sequence repositories, include, but are not limited to GenBank EMBL, DDBJ and the NCBI. Other repositories can easily be identified by searching on the internet. Polypeptides that can be produced using the methods described herein also include polypeptides have at least about 60%, 70%, 75%, 80%, 90%, 95%, or at least about 99% or more identity to any known or available
polypeptide (e.g., a therapeutic polypeptide, a diagnostic polypeptide, an industrial enzyme, or portion thereof, and the like). [00106] Purification moieties suitable for use with the methods described herein include, but are not limited to, polypeptides comprising one or more non-natural amino acids.
[00107] Purification moieties suitable for use with the methods described herein include, but are not limited to, cytokines, inflammatory molecules, growth factors, their receptors, and oncogene products or portions thereof. Examples of cytokines, inflammatory molecules, growth factors, their receptors, and oncogene products include, but are not limited to e.g., alpha- 1 antitrypsin, Angiostatin, Antihemo lytic factor, antibodies (including an antibody or a functional fragment or derivative thereof selected from: Fab, Fab', F(ab)2, Fd, Fv, ScFv, diabody, tribody, tetrabody, dimer, trimer or minibody), angiogenic molecules, angiostatic molecules, Apolipopolypeptide, Apopolypeptide, Asparaginase, Adenosine deaminase, Atrial natriuretic factor, Atrial natriuretic polypeptide, Atrial peptides, Angiotensin family members, Bone Morphogenic Polypeptide (BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP- 6, BMP-7, BMP-8a, BMP-8b, BMP-10, BMP-15, etc.); C-X-C chemokines (e.g., T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP- 10, GCP-2, NAP-4, SDF-1, PF4, MIG), Calcitonin, CC chemokines (e.g., Monocyte chemoattractant polypeptide- 1, Monocyte chemoattractant polypeptide-2, Monocyte chemoattractant polypeptide-3, Monocyte inflammatory
polypeptide-1 alpha, Monocyte inflammatory polypeptide- 1 beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262), CD40 ligand, C-kit Ligand, Ciliary
Neurotrophic Factor, Collagen, Colony stimulating factor (CSF), Complement factor 5a, Complement inhibitor, Complement receptor 1, cytokines, (e.g., epithelial Neutrophil Activating Peptide-78, GRO alpha/MGSA, GRO beta , GRO gamma , MIP-1 alpha , MIP-1 delta, MCP-1), deoxyribonucleic acids, Epidermal Growth Factor (EGF), Erythropoietin ("EPO", representing a preferred target for modification by the incorporation of one or more non-natural amino acid), Exfoliating toxins A and B, Factor IX, Factor VII, Factor VIII, Factor X, Fibroblast Growth Factor (FGF), Fibrinogen, Fibronectin, G-CSF, GM-CSF, Glucocerebrosidase, Gonadotropin, growth factors, Hedgehog polypeptides (e.g., Sonic, Indian, Desert), Hemoglobin, Hepatocyte Growth Factor (HGF), Hepatitis viruses, Hirudin, Human serum albumin, Hyalurin-CD44, Insulin, Insulin-like Growth Factor (IGF-I, IGF-II), interferons (e.g., interferon-alpha, interferon-beta, interferon-gamma, interferon-epsilon, interferon-zeta, interferon-eta, interferon-kappa, interferon-lambda, interferon-T, interferon- zeta, interferon-omega), glucagon-like peptide (GLP-1), GLP-2, GLP receptors, glucagon, other agonists of the GLP-1R, natriuretic peptides (ANP, BNP, and CNP), Fuzeon and other inhibitors of HIV fusion, Hurudin and related anticoagulant peptides, Prokineticins and related agonists including analogs of black mamba snake venom, TRAIL, RANK ligand and its antagonists, calcitonin, amylin and other glucoregulatory peptide hormones, and Fc fragments, exendins (including exendin-4), exendin receptors, interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, etc.), I-CAM- 1/LFA-l,
Keratinocyte Growth Factor (KGF), Lactoferrin, leukemia inhibitory factor, Luciferase, Neurturin, Neutrophil inhibitory factor (NIF), oncostatin M, Osteogenic polypeptide, Parathyroid hormone, PD-ECSF, PDGF, peptide hormones (e.g., Human Growth Hormone), Oncogene products (Mos, Rel, Ras, Raf, Met, etc.), Pleiotropin, Polypeptide A, Polypeptide G, Pyrogenic exotoxins A, B, and C, Relaxin, Renin, ribonucleic acids, SCF/c-kit, Signal transcriptional activators and suppressors (p53, Tat, Fos, Myc, Jun, Myb, etc.), Soluble complement receptor 1, Soluble I-CAM 1, Soluble interleukin receptors (IL-1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15), soluble adhesion molecules, Soluble TNF receptor, Somatomedin, Somatostatin, Somatotropin, Streptokinase, Superantigens, i.e., Staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE), Steroid hormone receptors (such as those for estrogen, progesterone, testosterone, aldosterone, LDL receptor ligand and corticosterone), Superoxide dismutase (SOD), Toll-like receptors (such as Flagellin), Toxic shock syndrome toxin (TSST-1), Thymosin a 1, Tissue plasminogen activator, transforming growth factor (TGF- alpha, TGF- beta), Tumor necrosis factor beta (TNF beta), Tumor necrosis factor receptor (TNFR), Tumor necrosis factor- alpha (TNF alpha), transcriptional modulators (for example, genes and transcriptional modular polypeptides that regulate cell growth, differentiation and/or cell regulation), Vascular Endothelial Growth Factor (VEGF), viruslike particle, VLA-4/VCAM-1, Urokinase, signal transduction molecules, estrogen, progesterone, testosterone, aldosterone, LDL, corticosterone.
[00108] Additional purification moieties suitable for use with the methods described herein include, but are not limited to, enzymes (e.g., industrial enzymes) or portions thereof.
Examples of enzymes include, but are not limited to amidases, amino acid racemases, acylases, dehalogenases, dioxygenases, diarylpropane peroxidases, epimerases, epoxide hydrolases, esterases, isomerases, kinases, glucose isomerases, glycosidases, glycosyl transferases, haloperoxidases, monooxygenases (e.g., p450s), lipases, lignin peroxidases, nitrile hydratases, nitrilases, proteases, phosphatases, subtilisins, transaminase, and nucleases. In certain embodiments, such enzymes comprising a PBRT or PBRC can be used as immobilized enzymes in industrial biocatalysis. The enzymes comprising a PBRTs or a PBRC can also be added to a solution to facilitate biocatalysis and then reisolated from the solution.
[00109] Additional purification moieties suitable for use with the methods described herein include, but are not limited to, agriculturally related polypeptides such as insect resistance polypeptides (e.g., Cry polypeptides), starch and lipid production enzymes, plant and insect toxins, toxin-resistance polypeptides, Mycotoxin detoxification polypeptides, plant growth enzymes (e.g., Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase), lipoxygenase, and Phosphoenolpyruvate carboxylase.
[00110] Additional purification moieties suitable for use with the methods described herein include, but are not limited to, antibodies, immunoglobulin domains of antibodies and their fragments. Examples of antibodies include, but are not limited to antibodies, antibody fragments, antibody derivatives, Fab fragments, Fab' fragments, F(ab)2 fragments, Fd fragments, Fv fragments, single-chain Fv fragments (scFv), diabodies, tribodies, tetrabodies, dimers, trimers, and minibodies.
[00111] Additional purification moieties suitable for use with the methods described herein include, but are not limited to, prophylactic vaccine or therapeutic vaccine polypeptides. A prophylactic vaccine is one administered to subjects who are not infected with a condition against which the vaccine is designed to protect. In certain embodiments, a preventive vaccine will prevent a virus from establishing an infection in a vaccinated subject. However, even if it does not provide complete protective immunity, a prophylactic vaccine may still confer some protection to a subject. For example, a prophylactic vaccine may decrease the symptoms, severity, and/or duration of the disease. A therapeutic vaccine, is administered to reduce the impact of a viral infection in subjects already infected with that virus. A
therapeutic vaccine may decrease the symptoms, severity, and/or duration of the disease. Vaccine polypeptides include polypeptides, or polypeptide fragments from infectious fungi (e.g., Aspergillus, Candida species) bacteria (e.g. E. coli, Staphylococci aureus)), or
Streptococci (e.g., pneumoniae); protozoa such as sporozoa (e.g., Plasmodia), rhizopods (e.g., Entamoeba) and flagellates (Trypanosoma, Leishmania, Trichomonas, Giardia, etc.); viruses such as (+) RNA viruses (examples include Poxviruses e.g., vaccinia; Picornaviruses, e.g., polio; Togaviruses, e.g., rubella; Flaviviruses, e.g., HCV; and Coronaviruses), (-) RNA viruses (e.g., Rhabdoviruses, e.g., VSV; Paramyxovimses, e.g., RSV; Orthomyxovimses, e.g., influenza; Bunyaviruses; and Arenaviruses), dsDNA viruses (Reoviruses, for example), RNA to DNA viruses, i.e., Retroviruses, e.g., HIV and HTLV, and certain DNA to RNA viruses such as Hepatitis B.
[00112] Additional purification moieties suitable for use with the methods described herein include, but are not limited to, molecules that comprise a chemical moiety selected from the group consisting of: cytotoxins, pharmaceutical drugs, dyes or fluorescent labels, a nucleophilic or electrophilic group, a ketone or aldehyde, azide or alkyne compounds, photocaged groups, tags, a peptide, a polypeptide, a polypeptide, an oligosaccharide, polyethylene glycol with any molecular weight and in any geometry, polyvinyl alcohol, metals, metal complexes, polyamines, imidizoles, carbohydrates, lipids, biopolymers, particles, solid supports, a polymer, a targeting agent, an affinity group, any agent to which a complementary reactive chemical group can be attached, biophysical or biochemical probes, isotypically-labeled probes, spin-label amino acids, fluorophores, aryl iodides and bromides.
[00113] Reference is also made to a "variant PBRT." A variant PBRT is a PBRT
comprising one or more amino acid substitutions any position in the sequence of SEQ ID NO: 1 wherein the substitution replaces any amino acid in position 1 through 9 with an amino acid having a similar side chain group, an amino acid having a similar side chain
configuration, an amino acid having an evolutionary positive relatedness, or an amino acid having an evolutionary neutral relatedness.
[00114] As used herein, the term "variant precipatable-beta roll cassette" (PBRC) refers to an amino acid sequence comprising at least one variant PBRT. In certain embodiments, a variant PBRC will comprise at least two variant PBRTs. In certain embodiments, a variant PBRC will comprise at least 3 variant PBRTs, at least 4 variant PBRTs, at least 5 variant PBRTs, at least 6 variant PBRTs, at least 7 variant PBRTs, at least 8 variant PBRTs, at least 9 variant PBRTs, at least 10 variant PBRTs, at least 11 variant PBRTs, at least 12 variant PBRTs, at least 13 variant PBRTs, at least 14 variant PBRTs, at least 15 variant PBRTs, at least 16 variant PBRTs, at least 17 variant PBRTs, at least 18 variant PBRTs, at least 19 variant PBRTs, at least 20 variant PBRTs, or 20 or more variant PBRTs. In certain embodiments, the PBRCs described herein will comprise a plurality of variant precipatable beta roll tags arranged in a tandem repeat. For example, in certain embodiments, the variant PBRCs described herein can comprise at least 2 variant PBRTs, at least 3 variant PBRTs, at least 4 variant PBRTs, at least 5 variant PBRTs, at least 6 variant PBRTs, at least 7 variant PBRTs, at least 8 variant PBRTs, at least 9 variant PBRTs, at least 10 variant PBRTs, at least 11 variant PBRTs, at least 12 variant PBRTs, at least 13 variant PBRTs, at least 14 variant PBRTs, at least 15 variant PBRTs, at least 16 variant PBRTs, at least 17 variant PBRTs, at least 18 variant PBRTs, at least 19 variant PBRTs, at least 20 variant PBRTs, or 20 or more variant PBRTs in tandem repeat. In certain embodiments, a PBRC can comprise at least two PBRCs separated by a linking amino acid sequence. Where a linking amino acid sequence in present between two PBRTs, a PBRTs located at either end of the linking sequence can be an individual PBRT or it can be a PBRTs that is part of a tandem arrangement.
[00115] Thus in certain embodiments, a variant PBRC will comprise at least 2 variant PBRCs, at least variant 3 PBRTs, at least 4 variant PBRTs, at least 5 variant PBRTs, at least 6 variant PBRTs, at least 7 variant PBRTs, at least 8 variant PBRTs, at least 9 variant PBRTs, at least 10 variant PBRTs, at least 11 variant PBRTs, at least 12 variant PBRTs, at least 13 variant PBRTs, at least 14 variant PBRTs, at least 15 variant PBRTs, at least 16 variant PBRTs, at least 17 variant PBRTs, at least 18 variant PBRTs, at least 19 variant PBRTs, at least 20 variant PBRTs, or 20 or more variant PBRTs, all of which are located N-terminally to a CS. In certain embodiments, a variant PBRC will comprise at least 2 variant PBRCs, at least variant 3 PBRTs, at least 4 variant PBRTs, at least 5 variant PBRTs, at least 6 variant PBRTs, at least 7 variant PBRTs, at least 8 variant PBRTs, at least 9 variant PBRTs, at least 10 variant PBRTs, at least 11 variant PBRTs, at least 12 variant PBRTs, at least 13 variant PBRTs, at least 14 variant PBRTs, at least 15 variant PBRTs, at least 16 variant PBRTs, at least 17 variant PBRTs, at least 18 variant PBRTs, at least 19 variant PBRTs, at least 20 variant PBRTs, or 20 or more variant PBRTs, all of which are located C-terminally to a CS.
[00116] In certain aspects, the invention relates to a variant PBRT that contains one or more amino acid insertions, deletions or substitutions as compared to the sequence of SEQ ID NO: 1 and wherein the variant PBRT retains an ability to bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the PBRT.
[00117] Changes can be introduced by mutation into nucleic acid sequences, thereby leading to changes in the amino acid sequence of the encoded protein, without altering the functional activity of a PBRT or a PBRC. For example, nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues can be made in the sequence of a PBRT or a PBRC. A non-essential amino acid residue is a residue that can be altered from the sequence of an amino acid of this invention without altering the ability of the PBRT or PBRC to bind to bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the PBRT or PBRC. [00118] Exemplary residues which are non-essential and therefore amenable to substitution to generate the variant PBRTs and PBRCs described herein can be identified by one of ordinary skill in the art by performing an amino acid alignment of two more PBRTs or PBRCs and determining residues that are not required for the PBRT or PBRC to bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the PBRT or PBRC.
[00119] Mutations can be introduced randomly along all or part of a nucleic acid sequence encoding a PBRT or PBRC, such as by saturation mutagenesis, and the resultant mutants can be screened, for example, for their ability bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT or variant PBRC. Following mutagenesis, the purification moiety linked to the variant PBRT or variant PBRC can be expressed recombinantly in a host cell and the functional activity of the precipatable beta-roll tag can be determined using assays available in the art for assessing binding to Ca2+, undergoing reversible precipitation in the presence of Ca2+, or inducing reversible precipitation of purification moiety linked to the variant PBRT or variant PBRC. In certain embodiments, the variant PBRTs described herein can comprise one or more amino acid substitutions, insertions or deletions, wherein the variant PBRT is functionally equivalent a PBRT having the sequence GGAGNDTLY (SEQ ID No. 1). In one embodiment, the variant PBRT has an identical ability bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety in a manner similar to, but not necessarily identical to a PBRC comprising only PBRTs of the sequence GGAGNDTLY. In one embodiment, the variant PBRT has a reduced ability bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety in a manner similar to, but not necessarily identical to a PBRC comprising only PBRTs of the sequence GGAGNDTLY. In one embodiment, the variant PBRT has an increased ability bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety in a manner similar to, but not necessarily identical to a PBRC comprising only PBRTs of the sequence GGAGNDTLY.
[00120] The variant PBRCs described herein can also comprise on or more PBRTs in addition to one or more variant PBRTs. The variant PBRCs described herein can also be employed in any embodiments or configuration described herein for a PBRC. Thus, the description of a composition comprising a PBRC, or a method comprising a PBRC applies equally to a variant PBRT or a variant PBRC so long as the variant PBRT or the variant PBRC can bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety in a manner similar to, but not necessarily identical to a PBRC comprising only PBRTs of the sequence GGAGNDTLY.
[00121] In one embodiment, a variant PBRT comprises the sequence GGXGXDXXX (SEQ ID NO: 2) wherein X can be selected from the group consisting of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. In one embodiment, the variant PBRT has a sequence of GGXGXDXXX (SEQ ID NO: 2), wherein X is not proline. In one embodiment, the variant PBRT comprises the sequence GGXGXDXXX (SEQ ID NO: 2) wherein X is a natural or non-natural amino acid comprising a modification.
[00122] In one embodiment, a variant PBRT or PBRC comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 98%, 99% identity with an amino acid sequence of SEQ ID NO: 1.
[00123] As used herein, "sequence identity" means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. Techniques for determining sequence identity are well known to one skilled in the art, and include, for example, analysis with a sequence comparison algorithm or FASTA version 3.0t78 using default parameters (Pearson and Lipman, Proc Natl Acad Sci U S A. 1988 Apr; 85(8):2444-8). In another non-limiting example, scoring of amino acid can be calculated using the PAM250 matrix as described in Dayhoff et al., (1978) in Atlas of Protein Sequence and Structure, ed. Dayhoff, M. (Natl. Biomed. Res. Found., Silver Spring, MD), Vol. 5, Suppl. 3, pp. 345-352.
[00124] Percent identity or percent similarity of a DNA or peptide sequence can be determined, for example, by comparing sequence information using the GAP computer program. The GAP program utilizes the alignment method of Needleman et al., 1970, as revised by Smith et al., 1981. Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred parameters for the GAP program are the default parameters, which do not impose a penalty for end gaps. See e.g., Schwartz et al., 1979; Gribskov et al., 1986. Nucleic acids that differ due to degeneracy of the genetic code, and still encode the PBRTs or PBRCs, described herein are encompassed by the present disclosure.
[00125] Variants can be produced by any number of methods, including but not limited to, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, and any combination thereof.
[00126] Variant PBRTs or variant PBRCs falling within the scope of this invention, can, in general, be generated by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the purification moiety at the target site, or (c) the bulk of the side chain.
[00127] In one embodiment, a variant PBRT or a variant PBRC can comprise a conservative amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain configuration. Amino acid residues having similar side chain configurations have been defined in the art within in accordance with the following categories: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine), and sulfur-containing side chains (methionine, cysteine). Substitutions can also be made between acidic amino acids and their respective amides (e.g., asparagine and aspartic acid, or glutamine and glutamic acid).
[00128] In one embodiment, a variant PBRT or a variant PBRC can comprise a conservative amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain group. Amino acid residues having similar side chain groups have been defined in the art within in accordance with the following categories: a no side chain group (glycine), an aliphatic side chain group (alanine, valine, leucine, isoleucine, proline), a hydroxyl side chain group (serine, threonine), an acidic side chain group (aspartic acid, glutamic acid), an amide side chain group (asparagine, glutamine), a basic side chain group (lysine, arginine), an imidazole side chain group (histidine), an aromatic side chain group (phenylalanine, tyrosine, tryptophan), and a sulfur containing side chain group
(methionine, cysteine) (see Sambrook et al, (2001) Molecular Cloning: A Laboratory Manual, Volume 3, Table A7-4).
[00129] In one embodiment, a variant PBRT or a variant PBRC can comprise a conservative amino acid substitution in which an amino acid residue is replaced an amino acid having evolutionarily positive relatedness. Amino acids having evolutionarily positive relatedness have been defined in the art as follows (wherein the amino acid(s) having evolutionarily positive relatedness are indicated in parentheses): Alanine (serine, threonine, proline, glycine); Arginine (glutamine, histidine, lysine, tryptophan); Asparagine (serine, threonine, aspartic acid, glutamic acid, glutamine, histidine, lysine); Aspartic acid (threonine, glycine, asparagine, glutamine, glutamic acid, histidine); Glutamic acid (threonine, asparagine, aspartic acid, glutamine, histidine); Glutamine (asparagine, aspartic acid, glutamic acid, histidine, arginine, lysine); Glycine (serine, threonine, alanine, aspartic acid); Histidine (asparagine, aspartic acid, glutamine, arginine); Isoleucine (threonine, methionine, leucine, valine, phenylalanine); Leucine (methionine, isoleucine, valine, phenylalanine); Lysine (threonine, asparagine, glutamine, arginine); Methionine (isoleucine, leucine, valine);
Phenylalanine (isoleucine, leucine, tyrosine); Proline (serine, threonine, alanine); Serine (threonine, proline, alanine, glycine, asparagine); Threonine (serine, proline, alanine, glycine, asparagine, aspartic acid, glutamic acid, lysine, isoleucine, valine); Tryptophan (arginine, tyrosine); Tyrosine (phenylalanine, tryptophan); Valine (threonine, methionine, isoleucine, leucine) (see Dayhoff et al., (1978) in Atlas of Protein Sequence and Structure, ed. Dayhoff, M., Natl. Biomed. Res. Found., Silver Spring, MD), Vol. 5, Suppl. 3, pp. 345-352).
[00130] In one embodiment, variant of a variant PBRT or a variant PBRC can comprise a conservative amino acid substitution in which an amino acid residue is replaced an amino acid having evolutionarily positive relatedness. Amino acids having evolutionarily neutral relatedness have been defined in the art as follows (wherein the amino acid(s) having evolutionarily neutral relatedness are indicated in parentheses): Alanine (asparagine, aspartic acid, glutamine, glutamic acid, valine); Arginine (serine, proline, asparagine, methionine); Asparagine (alanine, glycine, arginine); Aspartic acid (serine, alanine, lysine); Cysteine (serine, tyrosine); Glutamic acid (serine, alanine, glycine, lysine); Glutamine (proline, alanine); Glycine (asparagine, glutamic acid); Histidine (proline, lysine, tyrosine); Lysine (serine, asparagine, glutamic acid, histidine, methionine); Methionine (arginine, lysine, phenylalanine); Phenylalanine (methionine, tryptophan); Proline (glutamine, histidine, arginine); Serine (cysteine, aspartic acid, glutamic acid, arginine, lysine); Threonine (none); Tryptophan (phenylalanine); Tyrosine (cysteine, histidine); Valine (alanine) (see Dayhoff et al., (1978) in Atlas of Protein Sequence and Structure, ed. Dayhoff, M., Natl. Biomed. Res. Found., Silver Spring, MD), Vol. 5, Suppl. 3, pp. 345-352).
[00131] In one embodiment of a variant PBRT, the glycine at position 1 of SEQ ID NO: 1 is not mutated.
[00132] In another embodiment of a variant PBRT, the glycine at position 1 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the glycine is replaced with an amino acid having an uncharged polar side chain configuration (e.g., asparagine, glutamine, serine, threonine, tyrosine, or cysteine).
[00133] In another embodiment of a variant PBRT, the glycine at position 1 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the glycine is replaced with an amino acid having an aliphatic side chain configuration (e.g., alanine, valine, leucine, isoleucine)
[00134] In another embodiment of a variant PBRT, the glycine at position 1 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily positive relatedness such that the glycine is replaced with any of serine, threonine, alanine, or aspartic acid.
[00135] In another embodiment of a variant PBRT, the glycine at position 1 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily neutral relatedness such that the glycine is replaced with any of asparagine or glutamic acid.
[00136] In one embodiment of a variant PBRT, mutation of the glycine at position 1 of SEQ ID NO: 1 to any of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine wherein mutation of the glycine at position 1 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT. [00137] In one embodiment of a variant PBRT, mutation of the glycine at position 1 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid wherein mutation of the glycine at position 1 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
[00138] In one embodiment of a variant PBRT, the glycine at position 2 of SEQ ID NO: 1 can be mutated to an asparagine residue.
[00139] In one embodiment of a variant PBRT, the glycine at position 2 of SEQ ID NO: 1 can be mutated to an aspartic acid residue.
[00140] In another embodiment of a variant PBRT, the glycine at position 2 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the glycine is replaced with an amino acid having an uncharged polar side chain configuration (e.g., asparagine, glutamine, serine, threonine, tyrosine, or cysteine).
[00141] In another embodiment of a variant PBRT, the glycine at position 2 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the glycine is replaced with an amino acid having an aliphatic side chain configuration (e.g., alanine, valine, leucine, isoleucine)
[00142] In another embodiment of a variant PBRT, the glycine at position 2 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily positive relatedness such that the glycine is replaced with any of serine, threonine, alanine, or aspartic acid.
[00143] In another embodiment of a variant PBRT, the glycine at position 2 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily neutral relatedness such that the glycine is replaced with any of asparagine or glutamic acid.
[00144] In one embodiment of a variant PBRT, mutation of the glycine at position 2 of SEQ ID NO: 1 to any of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine wherein mutation of the glycine at position 2 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT. [00145] In one embodiment of a variant PBRT, mutation of the glycine at position 2 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid wherein mutation of the glycine at position 2 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
[00146] In one embodiment of a variant PBRT, the alanine at position 3 of SEQ ID NO: 1 can be mutated to a serine, glycine, or aspartic acid residue.
[00147] In one embodiment of a variant PBRT, the alanine at position 3 of SEQ ID NO: 1 can be mutated to a glutamic acid, leucine, or asparagine residue.
[00148] In one embodiment of a variant PBRT, the alanine at position 3 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the alanine is replaced with an amino acid having a nonpolar side chain configuration (e.g., valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan).
[00149] In one embodiment of a variant PBRT, the alanine at position 3 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the alanine is replaced with an amino acid having an aliphatic side chain configuration (e.g., glycine, valine, leucine, isoleucine)
[00150] In another embodiment of a variant PBRT, the alanine at position 3 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain group such that the alanine is replaced with an amino acid having an aliphatic side chain group (e.g., valine, leucine, isoleucine, proline).
[00151] In another embodiment of a variant PBRT, the alanine at position 3 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily positive relatedness such that the alanine is replaced with any of serine, threonine, proline, or glycine.
[00152] In another embodiment of a variant of a precipatable beta-roll tag, the at position 3 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily neutral relatedness such that the alanine is replaced with any of asparagine, aspartic acid, glutamine, glutamic acid, or valine.
[00153] In one embodiment of a variant PBRT, mutation of the alanine at position 3 of SEQ ID NO: 1 to any of glycine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine wherein mutation of the alanine at position 3 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
[00154] In one embodiment of a variant PBRT, mutation of the alanine at position 3 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid wherein mutation of the alanine at position 3 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
[00155] In one embodiment of a variant PBRT, the glycine at position 4 of SEQ ID NO: 1 can be mutated to an alanine residue.
[00156] In another embodiment of a variant PBRT, the glycine at position 4 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the glycine is replaced with an amino acid having an uncharged polar side chain configuration (e.g., asparagine, glutamine, serine, threonine, tyrosine, or cysteine).
[00157] In another embodiment of a variant PBRT, the glycine at position 4 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the glycine is replaced with an amino acid having an aliphatic side chain configuration (e.g., alanine, valine, leucine, isoleucine)
[00158] In another embodiment of a variant PBRT, the glycine at position 4 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily positive relatedness such that the glycine is replaced with any of serine, threonine, alanine, or aspartic acid.
[00159] In another embodiment of a variant PBRT, the glycine at position 4 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily neutral relatedness such that the glycine is replaced with any of asparagine or glutamic acid.
[00160] In one embodiment of a variant PBRT, mutation of the glycine at position 4 of SEQ ID NO: 1 to any of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine wherein mutation of the glycine at position 4 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
[00161] In one embodiment of a variant PBRT, mutation of the glycine at position 4 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid wherein mutation of the glycine at position 4 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
[00162] In one embodiment of a variant PBRT, the asparagine at position 5 of SEQ ID NO: 1 can be mutated to an aspartic acid or alanine residue.
[00163] In one embodiment of a variant PBRT, the asparagine at position 5 of SEQ ID NO: 1 can be mutated to a serine residue.
[00164] In one embodiment of a variant PBRT, the asparagine at position 5 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the asparagine is replaced with an amino acid having an uncharged polar side chain configuration (e.g., glycine, glutamine, serine, threonine, tyrosine, cysteine)
[00165] In one embodiment of a variant PBRT, the asparagine at position 5 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the asparagine is replaced with an amino acid having a the side chain configuration of its amide (e.g., aspartic acid).
[00166] In another embodiment of a variant PBRT, the asparagine at position 5 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain group such that the asparagine is replaced with an amino acid having an amide side chain group (e.g., glutamine).
[00167] In another embodiment of a variant PBRT, the asparagine at position 5 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily positive relatedness such that the asparagine is replaced with any of serine, threonine, aspartic acid, glutamic acid, glutamine, histidine, or lysine.
[00168] In another embodiment of a variant PBRT, the asparagine at position 5 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily neutral relatedness such that the asparagine is replaced with any of alanine, glycine, or arginine.
[00169] In one embodiment of a variant PBRT, mutation of the asparagine at position 5 of SEQ ID NO: 1 to any of glycine, alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine wherein mutation of the asparagine at position 5 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
[00170] In one embodiment of a variant PBRT, mutation of the asparagine at position 5 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid wherein mutation of the asparagine at position 5 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
[00171] In one embodiment of a variant PBRT, the aspartic acid at position 6 of SEQ ID NO: 1 can be mutated to an asparagine residue.
[00172] In one embodiment of a variant PBRT, the aspartic acid at position 6 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the aspartic acid is replaced with an amino acid having an acidic side chain configuration (e.g., glutamic acid).
[00173] In one embodiment of a variant PBRT, the aspartic acid at position 6 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the aspartic acid is replaced with an amino acid having a the side chain configuration of its amide (e.g., asparagine).
[00174] In another embodiment of a variant PBRT, the aspartic acid at position 6 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain group such that the aspartic acid is replaced with an amino acid having an acidic side chain group (e.g., glutamic acid).
[00175] In another embodiment of a variant PBRT, the aspartic acid at position 6 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily positive relatedness such that the aspartic acid is replaced with any of threonine, glycine, asparagine, glutamine, glutamic acid, or histidine.
[00176] In another embodiment of a variant PBRT, the aspartic acid at position 6 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily neutral relatedness such that the aspartic acid is replaced with any of serine, alanine, or lysine. [00177] In one embodiment of a variant PBRT, mutation of the aspartic acid at position 6 of SEQ ID NO: 1 to any of glycine, alanine, arginine, asparagine, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine wherein mutation of the aspartic acid at position 6 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
[00178] In one embodiment of a variant PBRT, mutation of the aspartic acid at position 6 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid wherein mutation of the aspartic acid at position 6 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
[00179] In one embodiment of a variant PBRT, the threonine at position 7 of SEQ ID NO: 1 can be mutated to an isoleucine or valine residue.
[00180] In one embodiment of a variant PBRT, the threonine at position 7 of SEQ ID NO: 1 can be mutated to a leucine residue.
[00181] In one embodiment of a variant PBRT, the threonine at position 7 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the threonine is replaced with an amino acid having an uncharged polar side chain configuration (e.g., glycine, asparagine, glutamine, serine, tyrosine, or cysteine).
[00182] In one embodiment of a variant PBRT, the threonine at position 7 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the threonine is replaced with an amino acid having a beta-branched side chain configuration (e.g., valine, isoleucine).
[00183] In another embodiment of a variant PBRT, the threonine at position 7 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain group such that the threonine is replaced with an amino acid having an hydroxyl side chain group (e.g., serine).
[00184] In another embodiment of a variant PBRT, the threonine at position 7 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily positive relatedness such that the threonine is replaced with any of serine, proline, alanine, glycine, asparagine, aspartic acid, glutamic acid, lysine, isoleucine, or valine [00185] In one embodiment of a variant PBRT, mutation of the threonine at position 7 of SEQ ID NO: 1 to any of glycine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, valine wherein mutation of the threonine at position 7 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
[00186] In one embodiment of a variant PBRT, mutation of the threonine at position 7 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid wherein mutation of the threonine at position 7 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
[00187] In one embodiment of a variant PBRT, the leucine at position 8 of SEQ ID NO: 1 can be mutated to an isoleucine residue.
[00188] In one embodiment of a variant PBRT, the leucine at position 8 of SEQ ID NO: 1 can be mutated to a phenylalanine residue.
[00189] In one embodiment of a variant PBRT, the leucine at position 8 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the leucine is replaced with an amino acid having a nonpolar side chain configuration (e.g., alanine, valine, isoleucine, proline, phenylalanine, methionine, or tryptophan).
[00190] In one embodiment of a variant PBRT, the leucine at position 8 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the leucine is replaced with an amino acid having an aliphatic side chain configuration (e.g., glycine, alanine, valine, or isoleucine).
[00191] In another embodiment of a variant PBRT, leucine at position 8 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain group such that the leucine is replaced with an amino acid having an aliphatic side chain group (e.g., alanine, valine, isoleucine, proline).
[00192] In another embodiment of a variant PBRT, the leucine at position 8 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily positive relatedness such that the leucine is replaced with any of methionine, isoleucine, valine, or phenylalanine [00193] In another embodiment of a variant PBRT, the leucine at position 8 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily neutral relatedness such that the leucine is replaced with any of serine, asparagine, glutamic acid, histidine, or methionine.
[00194] In one embodiment of a variant PBRT, mutation of the leucine at position 8 of SEQ ID NO: 1 to any of glycine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine wherein mutation of the leucine at position 8 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
[00195] In one embodiment of a variant PBRT, mutation of the leucine at position 8 of SEQ ID NO: 1 non-natural amino acid wherein mutation of leucine at position 8 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
[00196] In one embodiment of a variant PBRT, the tyrosine at position 9 of SEQ ID NO: 1 can be mutated to an isoleucine or valine residue.
[00197] In one embodiment of a variant PBRT, the tyrosine at position 9 of SEQ ID NO: 1 can be mutated to a phenylalanine, threonine, asparagine, aspartic acid, lysine, or serine residue.
[00198] In one embodiment of a variant PBRT, the tyrosine at position 9 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the tyrosine is replaced with an amino acid having an uncharged polar side chain configuration (e.g., glycine, asparagine, glutamine, serine, threonine, or cysteine).
[00199] In one embodiment of a variant PBRT, the tyrosine at position 9 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain configuration such that the tyrosine is replaced with an amino acid having an aromatic side chain configuration (e.g., tyrosine, phenylalanine, tryptophan, or histidine).
[00200] In another embodiment of a variant PBRT, the tyrosine at position 9 of SEQ ID NO: 1 can be mutated to an amino acid having a similar side chain group such that the tyrosine is replaced with an amino acid having an aromatic side chain group (e.g., phenylalanine, tryptophan).
[00201] In another embodiment of a variant PBRT, the tyrosine at position 9 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily positive relatedness such that the tyrosine is replaced with any of phenylalanine or tryptophan.
[00202] In another embodiment of a variant PBRT, the tyrosine at position 9 of SEQ ID NO: 1 can be mutated to an amino acid having evolutionarily neutral relatedness such that the tyrosine is replaced with any of cysteine or histidine.
[00203] In one embodiment of a variant PBRT, mutation of the tyrosine at position 9 of SEQ ID NO: 1 to any of glycine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine wherein mutation of tyrosine at position 9 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
[00204] In one embodiment of a variant PBRT, mutation of the tyrosine at position 9 of SEQ ID NO: 1 is with a non-natural or synthetic amino acid wherein mutation of the tyrosine at position 9 of SEQ ID NO: 1 will result in a precipatable beta-roll tag that is capable of binding to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the variant PBRT.
[00205] As described herein, a variant PBRC can further comprise a capping sequence. In certain embodiments, the capping sequence in a variant PBRC can be a variant capping sequence. A variant capping sequence can be an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identity to SEQ ID NO: 3. In another embodiment, a variant capping sequence is an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 98% identity to any of SEQ ID NO: 4-23.
[00206] In still a further embodiment, a variant capping sequence is a sequence comprising one or more amino acid substitutions with an amino acid having a similar side chain group. In one embodiment, the variant capping sequence comprises the sequence of SEQ ID NO: 3 or any of SEQ ID NO: 4-23, wherein at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, or at least 68, amino acids in the sequence of SEQ ID NO: 3 or any of SEQ ID NO: 4-23 are substituted an amino acid having a similar side chain group.
[00207] In still a further embodiment, a variant capping sequence is a sequence comprising one or more amino acid substitutions with an amino acid having a similar side chain configuration. In one embodiment, the variant capping sequence comprises the sequence of SEQ ID NO: 3 or any of SEQ ID NO: 6-23, wherein at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, or at least 68, amino acids in the sequence of SEQ ID NO: 3 or any of SEQ ID NO: 6-23 are substituted an amino acid having a similar side chain configuration.
[00208] In still a further embodiment, a variant capping sequence is a sequence comprising one or more amino acid substitutions with an amino acid having evolutionarily positive relatedness. In one embodiment, the variant capping sequence comprises the sequence of SEQ ID NO: 3 or any of SEQ ID NO: 6-23, wherein at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, or at least 68, amino acids in the sequence of SEQ ID NO: 3 or any of SEQ ID NO: 6-23 are substituted an amino acid having evolutionarily positive relatedness.
[00209] In still a further embodiment, a variant capping sequence is a sequence comprising one or more amino acid substitutions with an amino acid having evolutionarily neutral relatedness. In one embodiment, the variant capping sequence comprises the sequence of SEQ ID NO: 3 or any of SEQ ID NO: 6-23, wherein at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, or at least 68, amino acids in the sequence of SEQ ID NO: 3 or any of SEQ ID NO: 4-23 are substituted an amino acid having evolutionarily neutral relatedness.
[00210] In another embodiment, the PBRT variants or PBRC variants described herein can also comprise a non-natural amino acid. As used herein, a non-natural amino acid can be, but is not limited to, an amino acid comprising a moiety where a chemical moiety is attached, such as an aldehyde- or keto-derivatized amino acid, or a non-natural amino acid that includes a chemical moiety. A non-natural amino acid can also be an amino acid comprising a moiety where a saccharide moiety can be attached, or an amino acid that includes a saccharide moiety. Examples of non-classical amino acids suitable for use with the methods and compositions described herein include, but are not limited to, D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, gamma-Abu, epsilon-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,
phenylglycine, cyclohexylalanine, beta-alanine, fluoro-amino acids, designer amino acids such as beta-methyl amino acids, C alpha-methyl amino acids, N alpha-methyl amino acids, and amino acid analogs in general. [00211] The PBRT variants or PBRC variants described herein can also comprise one or more amino acid analog substitutions, e.g., unnatural amino acids such as alpha alpha- disubstituted amino acids, N-alkyl amino acids, lactic acid, and the like. These analogs include phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma- carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, 1,2,3,4,- tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citruline, . alpha. -methyl- alanine, para-benzoyl-phenylalanine, phenylglycine, propargylglycine, sarcosine, .epsilon.- Ν,Ν,Ν-trimethyllysine, .epsilon.-N-acetyllysine, N-acetylserine, N-formylmethionine, 3- methylhistidine, 5-hydroxylysine, . omega. -N-methylarginine, and other similar amino acids and imino acids and tert-butylglycine. The ability of PBRTs or PBRCs comprising an analog substitutions to bind to Ca2+, undergo reversible precipitation in the presence of Ca2+, or induce reversible precipitation of a purification moiety linked to the PBRT or PBRC using methods known to those skilled in the art.
[00212] The PBRT variants or PBRC variants described herein can further comprise polypeptide analogs, such as peptide mimetics (Fauchere J, Adv. Drug Res. 15:29 (1986); Veber D F and Freidinger R M, Trends Neurosci. 8:392-96 (1985); Evans B E et al, J. Med. Chem 30: 1229-39 (1987)). Generally, peptidomimetics are structurally similar to a template polypeptide (i.e., a polypeptide that has a biological or pharmacological activity), such as the PBRTs or PBRCs described herein, but have one or more peptide linkages replaced by a linkage selected from the group consisting of: ~CH.sub.2NH~, ~CH.sub.2S~,—CH.sub.2— CH.sub.2-, ~CH.dbd.CH~ (cis and trans), -COCH.sub.2-, -CH(OH)CH.sub.2-, and - CH.sub.2SO— , by methods known in the art and further described in the following references: Spatola A F in "Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins," B. Weinstein, ed., Marcel Dekker, New York, p. 267 (1983); Spatola, A F, Vega Data (March 1983), Vol. 1, Issue 3, "Peptide Backbone Modifications" (general review); Morley J S, Trends Pharmcol. Sci. 1 :463-68 (1980) (general review); Hudson D et al., Int. J. Pept. Prot. Res. 14: 177-85 (1979) (-CH.sub.2NH-, CH.sub.2CH.sub.2-); Spatola A F et al., Life Sci. 38: 1243-49 (1986) (-CH.sub.2-S); Hann M M, J. Chem. Soc. Perkin Trans. 1, 307- 314 (1982) (-CH-CH-, cis and trans); Almquist R G et al, J. Med. Chem. 23: 1392-98 (1980) (-COCH.sub.2-); Jennings- White C et al, Tetrahedron Lett. 23:2533-34 (1982) (- COCH.sub.2-); EP 0 045 665 (-CH(OH)CH.sub.2-); Holladay M W et al, Tetrahedron Lett., 24:4401-04 (1983) (-C(OH)CH.sub.2-); Hruby V J, Life Sci. 31 : 189-99 (1982) (- CH.sub.2— S— ). One example of a non-peptide linkage is—CH.sub.2NH-. [00213] Such polypeptide mimetics can have advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others. Labeling of peptidomimetics can involve covalent attachment of one or more labels, directly or through a spacer (e.g., an amide group), to non-interfering position(s) on the peptidomimetic that are predicted by quantitative structure-activity data and/or molecular modeling. Such non- interfering positions can be positions that do not from direct contacts with the
macromolecules(s) to which the peptidomimetic binds to produce the therapeutic effect. Derivatization (e.g., labeling) of peptidomimetics can be done without substantially interfering with the desired biological or pharmacological activity of the peptidomimetic. The ability of any peptidomimetics to polypeptides can be assayed for the ability to bind 1,4,- benzothiazepine or derivatives thereof using methods know to those skilled in the art.
[00214] Systematic substitution of one or more amino acids of the PBRTs or PBRCs described herein with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can be used to generate additional PBRT and PBRT variants.
[00215] The following methods can be used in connection with the embodiments of the invention.
EXAMPLES
[00216] Example 1 : Purification of PBRT or PBRC linked Purification moieties
[00217] 17 tandem repeats of the amino acid sequence GGAGNDTLY followed by a C- terminal "capping" sequence (the sequence is provided below) were fused to maltose binding protein. This construct was expressed in E. coli and the cells are lysed, creating a complex mixture of E. coli proteins and the Beta Roll tagged maltose binding protein. The mixture was exposed to 100 mM calcium chloride solution to form a precipitate form. The precipitate was pelleted and resuspended in calcium- free buffer. One precipitation cycle was sufficient to generate a relatively pure protein (Figure 1). Multiple cycles can be used to achieve better purity. SDS-PAGE samples of this procedure can be found in the supplemental materials.
[00218] The capping sequence used in this example is:
INAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQAGI EKLVEAMAQYPD (SEQ ID NO: 3) [00219] Polypeptides comprising a 5 or 17 repeat C-capped precipatable beta-roll tags were expressed as MBP fusion proteins. 50 mM calcium was added to clarified cell ly sates to induce precipitation of the polypeptides comprising a 5 or 17 repeat C-capped precipatable beta-roll tags. The precipitate was pelleted by centrifugation and the pellet was washed once and resuspended in buffer with 50 mM EGTA. The lysate, the supernatant after calcium addition, and the resuspended pellet were then run on an SDS-PAGE gel (Figure 2). The results show that the methods described herein can be used to rapidly purify polypeptides comprising a 5 or 17 repeat C-capped precipatable beta-roll tags. Precipitation of purification moieties comprising a precipatable beta-roll tag can be confirmed by circular dichroism analysis (Figure 3).
[00220] An intein domain can be coupled to the construct so that the cleavage reaction and subsequent second precipitation can be examined. Other proteins, in addition to maltose binding protein can be used with the purification protocols described herein.
[00221] Example 2: PBRT sequence heat map.
[00222] The heat map for the precipatable beta-roll tag sequences described herein was determined by using BLAST to find beta roll sequences similar to the metalloprotease of S. marcescens and then quantifying the frequency of amino acids at each of the nine positions after beta roll sequences were identified (Figure 4). Certain positions in the precipatable beta- roll tag are not highly variable (e.g. positions 1 , 2, 4, 6, and 8), whereas other positions, exhibit moderate conservation. Positions 7, 9 are highly variable and can be substituted with any natural or non-natural amino acid.
[00223] Example 3: Exemplary PBRCs
[00224] Below are the amino acid sequences of two exemplary PBRCs.
[00225] GGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYINA GADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQAGIEKL VEAMAQYPD (SEQ ID NO: 4)
[00226] GGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGA GNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGG AGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYGGAGNDTLYIN AGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQAGIEK LVEAMAQYPD (SEQ ID NO: 5) [00227] Example 4: Sequences of PBRT and PBRC peptides that do not induce precipitation in response to Ca2+
[00228] The following peptides are soluble in the presence of calcium.
GSARDDVLIGDAGANVLNGLADNDVLSGGAGDDVLLGDEGSDLLSGDAGNDDLFG GQGDDTYLFGVGYGHDTIYESGGGHDTIRGSARDDVLIGDAGANVLNGLADNDVLS GGAGDDVLLGDEGSDLLSGDAGNDDLFGGQGDDTYLFGVGYGHDTIYESGGGHDTI RINAGADQLWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQAG IEKLVEAMAQYPD (SEQ ID NO: 1339)
GSARDDVLIGDAGANVLNGLADNDVLSGGAGDDVLLGDEGSDLLSGDAGNDDLFG GQGDDT YLFG VG YGHDTI YE S GGGHDTIRGD AG AN VLNGL ADND VL S GG AGDD VL LGDEGSDLLSGDAGNDDLFGGQGDDTYLFGVGYGHDTIYESGGGHDTIR (SEQ ID NO: 1340)
GSARDDVLIGDAGANVLNGLADNDVLSGGAGDDVLLGDEGSDLLSGDAGNDDLFG GQGDDT YLFG VG YGHDTI YE S GGGHDTIRGD AG AN VLNGL ADND VL S GG AGDD VL LGDEGSDLLSGDAGNDDLFGGQGDDTYLFGVGYGHDTIYESGGGHDTIRINAGADQ LWFARQGNDLEIRILGTDDALTVHDWYRDADHRVEIIHAANQAVDQAGIEKLVEAM AQYPD (SEQ ID NO: 1341)
GGSGNDVIVGNAANNVLKGGAGNDVLFGGGGADELWGGAGKDIFV (SEQ ID NO: 1341)
GGSGNDVIVGNAANNVLKGGAGNDVLFGGGGADELWGGAGKDIFVFSAASDSAPG ASDWIRDFQKGIDKIDLSFFNKEANSSDFIHFVDHFSGTAGEALLSYNASSNVTDLSV NIGGHQAPDFLVKIVGQVDVATDFIV (SEQ ID NO: 1342)
[00229] References
[00230] Hochuli et al., "Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent", Nature Biotechnology, Issue 6, pp 1321 - 1325 (1988)
[00231] Guana et al., "Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein", Gene, Volume 67, Issue 1, pp 21-30, July 15 1988
[00232] Banki et al., "Simple bioseparations using self-cleaving elastin-like polypeptide tags", Nature Methods, Issue 2, pp 659 - 662, August 2, 2005 [00233] Wood et al., "A genetic system yields self-cleaving inteins for bioseparations." Nature Biotechnology, Issue 17, Pages 889 - 892 September 17, 1999.
[00234] U.S. Ser. No.: 11/374403

Claims

WHAT IS CLAIMED IS:
1. A precipatable beta roll cassette (PBRC) comprising one or more beta roll tags (PBRTs) wherein the one or more PBRTs comprise the amino acid sequence of SEQ ID NO: 1
2. A precipatable beta roll cassette (PBRC) comprising one or more beta roll tags (PBRTs) wherein the one or more PBRTs are independently any of:
(a) a polypeptide having the amino acid sequence of SEQ ID NO: 1, or
(b) a polypeptide having the amino acid sequence of any of SEQ ID NOs 25- 1337
3. A precipatable beta roll cassette (PBRC) comprising one or more beta roll tags (PBRTs) wherein the one or more PBRTs are independently any of:
(a) a polypeptide having the amino acid sequence of SEQ ID NO: 1, or
(b) a polypeptide having the amino acid sequence of any of SEQ ID NOs 25- 1337
(c) a polypeptide comprising the amino acid sequence GXXXXXXXX, wherein,
(i) the X at position 2 is an amino acid selected from the group consisting of glycine, asparagine or aspartic acid, and
(ii) the X at position 3 is an amino acid selected from the group consisting of alanine, serine, glycine, aspartic acid, glutamic acid, leucine or asparagine, and
(iii) the X at position 4 is an amino acid selected from the group consisting of glycine or alanine, and
(iv) the X at position 5 is an amino acid selected from the group consisting of asparagine, aspartic acid, alanine, or serine, and (v) the X at position 6 is an amino acid selected from the group
consisting of aspartic acid or asparagine,
(vi) the X at position 7 is an amino acid selected from the group consisting of threonine, isoleucine, valine, or leucine, and
(vii) the X at position 8 is an amino acid selected from the group consisting of leucine, isoleucine, or phenylalanine, and
(viii) the X at position 9 is an amino acid selected from the group consisting of tyrosine, isoleucine, valine, phenylalanine, threonine, asparagine, aspartic acid, lysine or serine.
4. A precipatable beta roll cassette (PBRC) comprising one or more beta roll tags (PBRTs) wherein the one or more PBRTs are independently any of:
(a) a polypeptide having the amino acid sequence of SEQ ID NO: 1, or
(b) a polypeptide having the amino acid sequence of any of SEQ ID NOs 25- 1337
(c) a polypeptide comprising the amino acid sequence GXXXXXXXX, wherein,
(i) the X at position 2 is an amino acid selected from the group consisting of glycine, asparagine or aspartic acid, and
(ii) the X at position 3 is an amino acid selected from the group consisting of alanine, serine, glycine, aspartic acid, glutamic acid, leucine or asparagine, and
(iii) the X at position 4 is an amino acid selected from the group consisting of glycine or alanine, and
(iv) the X at position 5 is an amino acid selected from the group consisting of asparagine, aspartic acid, alanine, or serine, and (v) the X at position 6 is an amino acid selected from the group
consisting of aspartic acid or asparagine,
(vi) the X at position 7 is an amino acid selected from the group consisting of threonine, isoleucine, valine, or leucine, and
(vii) the X at position 8 is an amino acid selected from the group consisting of leucine, isoleucine, or phenylalanine, and
(viii) the X at position 9 is an amino acid selected from the group consisting of tyrosine, isoleucine, valine, phenylalanine, threonine, asparagine, aspartic acid, lysine or serine, or
(c) a variant PBRT.
5. The PBRC of any of claims 1-4 further comprising a capping sequence.
6. The PBRC of any of claims 1-4 further comprising a stabilizing polypeptide.
7. A PBRC linked purification moiety comprising the PBRC of any of claims 1-4.
8. The PBRC linked purification moiety of claim 7 wherein the PBRC is linked to the purification moiety by a peptide bond.
9. The PBRC linked purification moiety of claim 7 wherein the PBRC is linked to the purification moiety by a chemical bond that is not a peptide bond.
10. The PBRC of any of claims 1-4 further comprising a cleavage site located N- terminally or C-terminally to one or more of the one or more PBRTs.
11. The PBRC of claim 10, wherein the cleavage site is selected from the group
comprising an intein cleavage site, a Factor Xa cleavage site, a thrombin cleavage site, an enterokinase cleavage site, or a signal peptidase cleavage site.
12. A polypeptide comprising the PBRC of any of claims 1-4 and a purification moiety.
13. A nucleic acid sequence encoding the polypeptide of any of claims 1-12.
14. A method for purifying a PBRC linked purification moiety, the method comprising (a) expressing the PBRC linked purification moiety in an expression system,
(b) collecting the PBRC linked purification moiety in a first medium,
(c) adding Ca2+ to the first medium so as to induce precipitation of PBRC linked purification moiety,
(d) removing unprecipiated material from the medium from the precipitated PBRC linked purification moiety,
(e) resuspending the PBRC linked purification moiety in a second medium having a lower than the free Ca2+ concentration than the free Ca2+ concentration obtained after step (c).
15. The method of claim 14 wherein a calcium chelator is added to the second medium of step (e).
16. The method of claim 14, wherein steps (c) to (e) are repeated one or more times.
17. The method of claim 14, further comprising a step of removing precipitated material between step (b) and step (c).
18. The method of claim 14, wherein the PBRC comprises a cleavage site between the PBRC and the purification moiety.
19. The method of claim 14, further comprising steps of:
(i) cleaving the PBRC linked purification moiety so as to separate the purification moiety from the PBRC,
(ii) adding Ca2+ to the medium so as to induce precipitation of the PBRC, and
(iii) isolating the unprecipiated purification moiety.
20. The method of claim 19, wherein the cleavage site is an intein cleavage site.
21. An expression vector comprising, as arranged from 5' to 3', a promoter, a nucleic acid sequence encoding the PBRC of any of claims 1-4, and at least one cloning site. An expression vector comprising, as arranged from 5' to 3', a promoter, at least one cloning site, and a nucleic acid sequence encoding the PBRC of any of claims 1-4.
An expression vector comprising, as arranged from 5' to 3', a promoter, at least one cloning site, a nucleic acid sequence encoding the PBRC of any of claims 1-4 and at least a second cloning site.
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