EP1115854A1 - Procede de production de permuteines par permutagenese d'exploration - Google Patents

Procede de production de permuteines par permutagenese d'exploration

Info

Publication number
EP1115854A1
EP1115854A1 EP99949630A EP99949630A EP1115854A1 EP 1115854 A1 EP1115854 A1 EP 1115854A1 EP 99949630 A EP99949630 A EP 99949630A EP 99949630 A EP99949630 A EP 99949630A EP 1115854 A1 EP1115854 A1 EP 1115854A1
Authority
EP
European Patent Office
Prior art keywords
cag
ctg
tct
protein
amino acid
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
Application number
EP99949630A
Other languages
German (de)
English (en)
Inventor
Stephen C. Lee
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GD Searle LLC
Original Assignee
GD Searle LLC
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by GD Searle LLC filed Critical GD Searle LLC
Publication of EP1115854A1 publication Critical patent/EP1115854A1/fr
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • a method of producing circularly-permuted proteins (permuteins) by scanning permutagenesis comprises making and inserting a series of circularly- permuted genes into a display vector, expressing these genes such that the gene products are localized to the surface of the display vector, generating a library of display vectors presenting the permuted protein, affinity-selecting the display vectors with a target protein that can bind the permuted protein, isolating and analyzing clones of selected display vectors to identify the circularly-permuted protein.
  • the invention further discloses methods of expressing and uses of permuteins.
  • Circularly permuted proteins are made by reordering the primary sequence of a parent protein.
  • the amino and carboxy terminal ends of the parent protein are joined by a peptide linker and new amino and carboxy terminal ends are generated at other positions in the sequence. This technique of generating variants has been applied to a wide variety of proteins (Table 1).
  • Circularly permuted proteins in many cases, are structurally and functionally similar to their non-permuted parent molecule after they undergo refolding.
  • the information necessary to direct the folding of proteins into tertiary structures is present in secondary structural domains. Vectorial folding of proteins from their native amino to carboxy ends is not often observed.
  • the ability of permuteins to retain structural and functional properties is remarkable, extending earlier observations on the plasticity of proteins with respect to amino acid substitutions (Olins P.O. et al., J. Biol. Chem. 270: 23754-23760, 1995; Lowman and Wells, J. Mol. Biol. 234: 564-578, 1993) and short amino acid insertions (Sondek, J. and D. Shortle, Proteins 7: 387-393, 1990; Shortle, D. and J. Sondek, Curr. Opin. Biotechno 6: 299-305).
  • proteins which range in size from 58 to 462 amino acids (Goldenberg & Creighton, J. Mol. Biol. 165:407- 413, 1983: Li & Coffino, Mol. Cell. Biol. 13:2377-2383, 1993).
  • the proteins represent a broad range of structural classes, including proteins that contain predominantly alpha helix (interleukin-4; Kreit an et al., Cytokine 7:311-318, 1995), beta sheet (inte ⁇ leukin-1; Horlick et al., Protein Eng. 5:427-431.
  • yeast phosphoribosyl anthranilate isomerase yeast phosphoribosyl anthranilate isomerase
  • Luger et al. Science 243:2,06-210, 1989
  • broad categories of protein function are represented in these sequence reorganization studies, the results of these studies have been highly variable. In many cases substantially lower activity, solubility, or thermodynamic stability were observed (E. coli dihydrofolate reductase, aspartate transcarbamoylase, phosphoribosyl anthranilate isomerase, glyceraldehyde-3- phosphate dehydrogenase, ornithine decarboxylase, ompA, yeast phosphoglycerate dehydrogenase).
  • sequence rearranged protein appeared to have ⁇ " many nearly identical properties as its natural counterpart (basic pancreatic trypsin inhibitor, T4 lysozyme, ribonuclease Tl, Bacillus ⁇ -glucanase, interleukin- l ⁇ , ⁇ -spectrin SH3 domain, pepsinogen, interleukin-4).
  • Protein permutagenesis can be used to optimize the activity of fusion proteins or proteins conjugated to other molecules.
  • a fusion between interleukin-4 _ (IL-4) and Pseudomonas exotoxin has been permuted resulting in a protein that has the first amino acid of the IL-4 domain at position 38 and the new carboxy end occurs at amino acid position 37 (Kreitman, R. J. et al., Proc Natl Acad Sci USA 91: 6889-6893, 1994).
  • the permuted fusion has increased affinity for the IL-4 receptor, increased cytotoxicity to IL-4 receptor bearing renal carcinoma cells, and increased anti-tumor activity in a murine model, compared to the non-permuted parent fusion protein (Kreitman, R. J. et al., Proc Natl Acad Sci USA 91: 6889-6893, 1994; Kreitman, R. J. et al., Cancer Res. 55:3357-3363, 1995; Puri, R. K. et al., Cellular Immunol. 171: 80-86, 1996). Increased potency of the permuted molecule is believed to result from a reduction in steric interference between the IL-4 domain in the parent molecule and its receptor.
  • affinity isolation of the presented protein also leads to affinity isolation of the corresponding genes.
  • Extremely large libraries of phage presented proteins are constructed and affinity screened very rapidly. From the standpoint of how quickly mutant proteins can be made and screened for activity, phage display is the most efficient mutagenesis technique currently available.
  • Permuteins can have improved biological properties by acting through several mechanisms.
  • the permutein acting on the same type of cell as its parent molecule may have increased binding, or other action, by virtue of increased avidity.
  • Dimers or higher order multimers of these proteins with themselves or other chemical groups, including proteins, can have increased efficacy or potency, or both.
  • Permuteins can also have improved therapeutic properties through a variety of mechanisms such as: (1) alterations in the overall on- or off-rates or K a or K d of the ligand(s) on the target cell; (2) activation or blockade of complementary receptor signaling pathways; and/or (3) more specific targeting of to the cell of interest.
  • the permuteins may also possess a unique pharmacokinetic distribution and clearance profile (Deh er et al., Circulation, 91, 2188-2194, 1995; Tanaka et al.,. Nature Medicine, 3, 437-442, 1997).
  • Permuteins can also have improved properties in vivo, compared to the two components individually, as a result of alterations in biodistribution or half-life.
  • the improved properties can also result from the binding of the permutein to one or more of the receptors, pharmacokinetics, or uptake of the permutein is altered in a favorable manner.
  • the present invention is an improved method for generating permuteins
  • Phage display is a powerful, yet convenient tool, traditionally used for mutagenesis and screening (Clackson, T. and J.A. Wells, Tibtech 12: 173-
  • the present invention relates to methods of producing biologically-active circularly permuted proteins of the formula C'-L'-N 1 , derived from a parent protein of the formula N'-C, wherein C 1 is comprised of a segment derived from the carboxy portion of said parent protein; N 1 is comprised of a segment derived from the amino terminal portion of said parent protein; and L 1 is a chemical bond or a linker, linking C to the amino terminus of L 1 and carboxy terminus of L 1 to the amino terminus of N 1 ; comprising the steps of: (a) making a series of circularly- permuted genes; (b) inserting said circularly-permuted genes into a display vector; (c) expressing said circularly-permuted genes such that the proteins encoded by said genes are presented on the surface of the display vector; (d) generating a library of display vectors presenting the expressed circularly permuted protein; (e) affinity-select the presenting display vectors with a target protein that
  • the method of making a series of circularly-permuted genes is selected from the group consisting of making a tandemly-repeated intermediate, total synthesis of a synthetic gene, assembly of a gene from synthetic oligonucleotides, DNA amplification, and limited digestion of a circular intermediate.
  • the display vector is selected from the group consisting of bacteriophage display vectors, bacteria, and baculovirus vectors. Even more preferably the presentation vector is a bacteriophage. Even more preferably, the presentation vector is bacteriophage M13. Most preferably, the presentation vector is a bacteriophage M13 gene III vector.
  • the method of making a series of circularly permuted genes is a method of making a tandem repeat intermediate. Even more preferably circularly permuted genes are amplified from the repeat by gene amplification.
  • the method of affinity selection comprises the steps consisting of (a) binding said presentation display vectors to a target protein; (b) eluting said display vectors; (c) amplifying said display vectors; and (d) biopanning a pool of _ said amplified display vectors.
  • the length of C in the permutein is longer than the length of C 1 in said parent protein. More preferably, the length of C in the permutein is shorter than the length of C in said parent protein. Most preferably, the length of
  • C in the permutein is the same length as the length of C in said parent protein.
  • the length of N 1 in the permutein is longer than the length of N l in said parent protein. More preferably, the length of N 1 in the permutein is shorter than the length of N 1 in said parent protein. Most preferably, the length of N 1 in the permutein is the same length as the length of N 1 in said parent protein.
  • the invention also contemplates circularly permuted proteins of the formula C'-L'-N 1 made by the method of scanning permutagenesis.
  • the DNA sequence encoding said linker L 1 is selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 368.
  • the circularly-permuted protein is the G-CSF receptor agonist domain of a species of mylepoietin (MPO).
  • MPO is one member of a family of novel dual cytokine receptor agonists (McKearn, J.P., Myelorestorative activities of synthokine and myelopoietin. In Proceedings of the 1996 IBC Conference on Therapeutic Applications of Cytokines, pp. 4.3.1-4.3.18, 1996) which are amenable to manipulation by phage display (Merlin, S. et al., Applied Biochemistry and
  • Plate A of Figure 1 shows the strategy to generate a scanning permutagenesis phage display library.
  • a plasmid containing directly-repeated tandem copies of the hG-CSF gene for example, is constructed by standard methods.
  • the tandem repeat plasmid is used as the template for PCR amplification of genes encoding permuted proteins.
  • Each copy of the G-CSF gene is indicated in light gray (turquoise), and a DNA segment encoding a peptide linker is indicated in dark gray (red).
  • oligonucleotide primers that initiate PCR polymerization at the first nucleotide of a chosen codon of G-CSF, and directing polymerization to the end of the tandem construct specifying the carboxy end of the protein encoded on the template is annealed to the tandem template.
  • a second specific primer is also annealed to the template that initiates polymerization at the last nucleotide of the codon encoding the amino acid immediately preceding the codon where polymerization begins with first primer, and which directs polymerization in the opposite direction from that first primer. Amplification between these two primers produces a DNA segment encoding a permuted protein.
  • amplification between the primer indicated by a black arrow initiating at codon 2 and the primer indicated by the blue arrow and initiating at the codon before 2 produces an amplified gene encoding a permuted protein whose amino terminal residue is amino acid 2 of the native protein, and whose final amino acid is amino acid 1 of the native protein.
  • a linker peptide is present between the first and final amino acids of the parent protein (residues 1 and 174 in this example).
  • a total of 174 individual amplifications would produce a complete collection of all permuted proteins of this example. More limited collections containing only a selected set of permuteins can be made, as well as more extensive collections made from multiple tandem template plasmids, each containing a different linker sequence between the first and last residues of the two directly repeated tandem gene sequences.
  • the collection of amplified segments can then be inserted into a phagemid presentation vector by standard methods. Phagemid particles produced from these presentation constructs are the scanning permutagenesis phage display library.
  • Plate B of Figure 1 shows the affinity screening of a phage display library
  • a hG-CSF scanning permutagenesis library as described in Figure 1A is screened using the hG-CSF receptor expressed on mammalian cells as the affinity reagent.
  • individual presented proteins are indicated by the shaded circles or diamonds and the affinity reagent is indicated by the light gray (pink) rectangles.
  • Presentation library particles are exposed to affinity reagent, unbound particles are washed away, and receptor-bound particles are eluted. The eluted particles are amplified in E. coli, and the screening cycle is repeated. During any round of the screening cycle, the genes encoded (in the present example encoding permuted proteins) by the selected particles can be expressed and evaluated.
  • Human G-CSF (serl7) protein is depicted as a string of circles, each circle corresponding to a single amino acid residue. Amino and carboxy ends of the protein are indicated. The amino acids of helical regions are indicated by medium gray balls, while the amino acids of inter-helical loops are indicated in light gray balls (See Hill et al., Proc. Natl. Acad. Sci. USA 90: 5167-5171, 1993). Amino ends of the permuteins made for presentation in the library are indicated in dark gray. Asterisks indicate the breakpoints of the presented permuteins which were isolated by affinity screening with cells expressing hG-CSF receptor as illustrated in IB.
  • Permeation molecules in the culture supernatants were quantitated by ELISA, and the proliferative activity of clones was determined using BAF-3-cells dependent on
  • G-CSF for growth.
  • the horizontal axis indicate concentration of protein and the vertical axis indicate incorporation of tritiated thymidine.
  • permutein means a circularly-permuted protein: a protein in which the amino and carboxy ends of the parent protein are joined together by a peptide linker sequence of zero or more amino acids. The amino and carboxy ends of the permuted protein occur at amino acids within the parental sequence.
  • chemical ligation and “conjugation” mean a chemical reaction which covalently links two similar or dissimilar functional groups together _ intramolecularly or intermolecularly.
  • peptide linker means a compound which forms a carboxamide bond between two groups having one or more peptide linkages (CONH-) and serves as a connector for the propose of amelioration of the distance or space orientation between two molecules.
  • mutant sequence refers to an amino acid or nucleic acid sequence which is identical to a wild-type or native form of a gene or protein.
  • mutant amino acid sequence refers to a polypeptide having an amino acid sequence which varies from a native sequence due to amino acid additions, deletions, substitutions, or all three, or is encoded by a nucleotide sequence from an intentionally-made variant derived from a native sequence.
  • the present invention encompasses circularly permuted-proteins of the formula C'-L'-N 1 prepared by phage display techniques.
  • the polypeptide can be joined either directly or through a linker segment.
  • the term "directly” defines permuteins in which the polypeptide ends are joined without a linker.
  • L 1 represents a chemical bond or a linker, preferably a polypeptide segment to which both C 1 and N 1 are joined, wherein C 1 is comprised of a segment derived from the carboxy portion of the parent protein and N 1 is comprised of a segment derived from the amino terminal portion of a parent protein represented by the general formula N'-C 1 .
  • N 1 and C 1 in the permuted protein C'-L'-N 1 are the same length as in the parent protein N'-C, but each may be independently shorter or longer depending on the desired structural characteristics of the permutein.
  • L' is a linear peptide in which C and N' are joined by amide bonds, linking C to the amino terminus of L' and carboxy terminus of L' to the amino terminus of N 1 .
  • Additional peptide sequences may also be added to facilitate purification or identification of permuteins (e.g., poly-His).
  • a highly antigenic peptide may also be added that would enable rapid assay and facile purification of the permuteins by a specific monoclonal antibody.
  • the linking group (L 1 ) is generally a polypeptide of between 1 and 500 amino acids in length.
  • the linkers joining the two molecules are preferably designed to (1) allow the two molecules to fold and act independently of each other,
  • surface amino acids in flexible protein regions include Gly, Asn and Ser. Virtually any permutation of amino acid sequences containing Gly, Asn and Ser would be expected to satisfy the above criteria for a linker sequence.
  • Other neutral amino acids such as Thr and Ala, may also be used in the linker sequence.
  • Additional amino acids may also be included in the linkers due to the addition of unique restriction sites in the linker sequence to facilitate construction of the multi-functional proteins.
  • Preferred L 1 linkers of the present invention include sequences selected from the group of formulas: (SEQ ID NO : 1 ) through SEQ ID NO : 2 68 )
  • linkers are also contemplated by the invention.
  • the present invention is, however, not limited by the form, size or number of linker sequences employed.
  • the only requirement of the linker is that it does not functionally interfere with the folding and function of the individual molecules of the multi- functional protein.
  • Permuteins of the present invention may exhibit useful properties such as having similar or greater biological activity when compared to a single factor or by having improved half-life or decreased adverse side effects, or a combination of these properties.
  • Permuteins which have little or no activity maybe useful as antigens for the production of antibodies for use in immunology or immunotherapy, as probes or as intermediates used to construct other useful permuteins.
  • the permuteins of the present invention may have an improved therapeutic profile as compared to their parent molecules.
  • some permuteins of the present invention may have a similar or more potent activities relative to other compounds or proteins without having a similar or corresponding increase in side-effects. This is particularly true of multifunctional or fusion protein therapeutics, where permutation may relieve steric and other hindrances that impair the activity of the parent fusion molecules (see Kreitman, R. J. et al., Proc Natl Acad Sci USA 91: 6889-6893, 1994; Kreitman, R. J. et al., Cancer Res. 55:3357-3363, 1995, for examples).
  • Nanoscale devices A general utility of permuteins is in the area of nanoscale devices described alternatively as “nanobiological” or “nanobiotechnological.” These are nanoscale devices containing both precise structure nanomaterials and biological functional components (such as proteins). Nanodevices have been the subject of several reviews (Lee, S.C., Trends in Biotechnology, 16: 239-240, 1998).
  • Nanobiological/nanobiotechnological devices generally contain proteins covalently coupled to polymers or other non-biological precise structure materials. Issues of steric and other interferences with protein activity are applicable to proteins in nanobiological/nanobiotechnological devices and are highly analogous to the issues with multifunctional/fusion proteins discussed above. Protein permutation is fully expected to offer a viable approach to deal with these considerations, just as it does in the case of fusion proteins (Kreitman, R. J. et al., Proc Natl Acad Sci USA 91: 6889-6893, 1994; Kreitman, R. J. et al.,., Cancer Res. 55:3357-3363, 1995).
  • Plasmids and bacteriophage used or constructed in this study are listed in Tables 2 and 3, respectively.
  • Phage and phagemid stocks were made and manipulated as described (Kay, B.K., Winter, J., and McCafferty, J., Phage Display of Peptides and Proteins, Academic Press, San Diego, California, 1996; Merlin, S. et al., Applied
  • E. coli strains such as DH5 ⁇ TM (Life Technologies, Gaithersburg, MD) and TGI (Amersham Corp., Arlington Heights, IL) are used for transformation of ligation reactions and are the hosts used to prepare plasmid
  • E. coli strains such as JM101 (Yanisch- Perron et al., Gene, 33: 103-119, 1985) and MON105 (Obukowicz et al., Appl and En ⁇ ir. Micr., 58: 1511-1523, 1992) can be used for expressing the multi-functional proteins of the present invention in the cytoplasm or periplasmic space.
  • DH5 ⁇ TM Subcloning efficiency cells are purchased as competent cells and are ready for transformation using the manufacturer ' s protocol, while both E. coli strains TGI and MON105 are rendered competent to take up DNA usinp a CaCl 2 method.
  • 20 to 50 mL of cells are grown in LB medium (1% Bacto- tryptone, 0.5% Bacto-yeast extract, 150 mM NaCl) to a density of approximately 1.0 optical density unit at 600 nanometers (OD600) as measured by a Baush & Lomb Spectronic spectrophotometer (Rochester, NY).
  • the cells are collected by centrifugation and resuspended in one-fifth culture volume of CaCl 2 solution (50 mM CaCl 2 , 10 mM Tris-Cl, pH7.4) and are held at 4°C for 30 minutes.
  • the cells are again collected by centrifugation and resuspended in one-tenth culture volume of - CaCl 2 solution.
  • Ligated DNA is added to 0.2 mL of these cells, and the samples are held at 4°C for 30-60 minutes.
  • the samples are shifted to 42°C for two minutes and 1.0 mL of LB is added prior to shaking the samples at 37°C for one hour.
  • Cells from these samples are spread on plates (LB medium plus 1.5% Bacto-agar) containing either ampicillin (100 micrograms/mL, ug/mL) when selecting for ampicillin-resistant transformants, or spectinomycin (75 ug/mL) when selecting for spectinomycin-resistant transformants. The plates are incubated overnight at 37°C.
  • Colonies are picked and inoculated into LB plus appropriate antibiotic (100 ug/mL ampicillin or 75 ug/mL spectinomycin) and are grown at 37°C while shaking.
  • appropriate antibiotic 100 ug/mL ampicillin or 75 ug/mL spectinomycin
  • DNA constructs were made and propagated in E. coli using standard molecular biology techniques (Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory, 1989).
  • Plasmid DNA can be isolated by a number of different methods and using commercially available kits known to those skilled in the art. Plasmid DNA is isolated using the Promega WizardTM Miniprep kit (Madison, Wl), the Qiagen
  • QIAwelt Plasmid isolation kits (Chatsworth, CA) or Qiagen Plasmid Midi or Mini kit. These kits follow the same general procedure for plasmid DNA isolation. Briefly, cells are pelleted by centrifugation (5000 x g), the plasmid DNA released with, sequential NaOH/acid treatment, and cellular debris is removed by centrifugation ( 10000 x g). The supernatant (containing the plasmid DNA) is loaded onto a column containing a DNA-binding resin, the column is washed, and plasmid DNA eluted. After screening for the colonies with the plasmid of interest, the E.
  • coli cells are inoculated into 50-100 ml of LB plus appropriate antibiotic for overnight growth at 37°C in an air incubator while shaking.
  • the purified plasmid DNA is used for DNA sequencing, further restriction enzyme digestion, additional subcloning of DNA fragments and transfection into E. coli, mammalian cells, or other cell types.
  • plasmid DNA is resuspended in dH 2 0 and its concentration is determined by measuring the absorbance at 260/280 nm in a Bausch and Lomb Spectronic 601 UV spectrometer. DNA samples are sequenced using ABI PRISMTM DyeDeoxyTM terminator sequencing chemistry (Applied Biosystems Division of
  • Perkin Elmer Corporation, Lincoln City, CA) kits Part Number 401388 or 402078, according to the manufacturer's suggested protocol usually modified by the addition of 5% DMSO to the sequencing mixture.
  • Sequencing reactions are performed in a DNA thermal cycler (Perkin Elmer Corporation, Norwalk, CT) following the recommended amplification conditions. Samples are purified to remove excess dye terminators with Centri-SepTM spin columns (Princeton Separations, Adelphia, NJ) and lyophilized. Fluorescent dye labeled sequencing reactions are resuspended in deionized formamide, and sequenced on denaturing 4.75% polyacrylamide-8M urea gels using ABI Model 373A and Model 377 automated DNA sequencers. Overlapping DNA sequence fragments are analyzed and assembled into master DNA contigs using Sequencher DNA analysis software (Gene Codes Corporation, Ann Arbor, MI).
  • DNA segments containing individual affinity-selected MPO: cphGCSFs were subcloned into a mammalian expression vector, and expressed transiently in BHK cells as described below.
  • the BHK-21 cell line can be obtained from the ATCC (Rockville, MD). The cells are cultured in Dulbecco's modified Eagle media (DMEM high-glucose), supplemented to 2 mM (mM) L-riutamine and 10% fetal bovine serum (FBS). This formulation is designated BHK growth media. Selective media is BHK growth media supplemented with 453 units/mL hygromycin B (CalBiochem, San Diego, CA).
  • DMEM high-glucose Dulbecco's modified Eagle media
  • FBS fetal bovine serum
  • the BHK-21 cell line was previously stably transfected with the HSV transactivating protein VP16, which transactiva es the IE110 promoter found on the plasmid pMON3359 and pMON3633 and the IE 175 promoter found in the plasmid pMON3360B (Hippenmeyer, P.J. and Pegg, L.E., Curr. Opin. Biotechnol. 6: 548-552, 1995).
  • the VP16 protein drives expression of genes inserted behind the IE110 or IE175 promoter.
  • BHK-21 cells expressing the transactivating protein VP16 are designated BHK-VP16.
  • the plasmid pMON1118 expresses the hygromycin resistance gene from the SV40 promoter (Highkin et al., Poultry Sci., 70: 970-981, 1991).
  • a similar plasmid, pSV2-hph, is available from ATCC.
  • BHK-VP16 cells are seeded into a 60 millimeter (mm) tissue culture dish at 3 x 10 5 cells per dish 24 hours prior to transfection.
  • Cells are transfected for 16 hours in 3 mL of "OPTIMEM”TM (Gibco-BRL, Gaithersburg, MD) containing 10 ug of plasmid DNA containing the gene of interest, 3 ug hygromycin resistance plasmid, pMONlll ⁇ , and 80 ug of Gibco-BRL "LIPOFECTAMINE”TM per dish.
  • the media is subsequently aspirated and replaced with 3 mL of growth media.
  • media from each dish is collected and assayed for activity (transient conditioned media).
  • the cells are removed from the dish by trypsin-EDTA, diluted 1:10, and transferred to 100 mm tissue culture dishes containing 10 mL of selective media. After approximately 7 days in selective media, resistant cells grow into colonies several millimeters in diameter. The colonies are removed from the dish with filter paper (cut to approximately the same size as the colonies and soaked in trypsin/EDTA) and transferred to individual wells of a 24 well plate containing 1 mL of selective media. After the clones are grown to confluence, the conditioned media is re-assayed, and positive clones are expanded into growth media.
  • Affinity reagent used for the identification of functional MPO molecules containing cphG-CSF (MPO: cphG-CSF) species from the library were BHK cells expressing the hG-CSF receptor on their surface.
  • the library pool was subjected to iterative affinity selection (four rounds) against BHK cells expressing the h-GCSF receptor using previously described techniques (Merlin, S. et al., Applied Biochemistry and Biotechnology 67: 15-29, 1997). Between rounds of selection, phage eluted from the affinity reagent were amplified in E. coli (Kay, B.K. J. Winter, and J. McCofferty, Phage Display of Peptides and Proteins, Academic Press, San Diego, California. 1996). Expression of proteins in E. coli
  • DNA segments containing individual affinity-selected MPO phGCSFs are subcloned into any of a variety of bacterial plasmid expression vectors, and expressed as a cytoplasmic product or as a secreted protein in E. coli.
  • E. coli strain MON105 or JM101 harboring the plasmid of interest are grown at 37°C in M9 plus casamino acids medium with shaking in an air incubator Model G25 from New Brunswick Scientific (Edison, NJ). Growth is monitored at OD gr ⁇ until it reaches a value of 1.0 at which time nalidixic acid (10 mg/mL) in 0.1 N NaOH is added to a final concentration of 50 ⁇ g/mL, for cultures containing plasmids with the E. coli recA promoter driving expression of the recombinant gene. IPTG is used in place of nalidixic acid, as a chemical inducer to facilitate expression from plasmids containing the lac promoter or hybrid lac promoters.
  • the cultures are then shaken at 37°C for three to four additional hours. A high degree of aeration is maintained throughout the culture period in order to achieve maximal production of the desired gene product.
  • the cells are examined under a light microscope for the presence of inclusion bodies (IB).
  • IB inclusion bodies
  • One mL aliquots of the culture are removed for analysis of protein content by boiling the pelleted cells, treating them with reducing buffer and electrophoresis via SDS-PAGE (see Maniatis et al., "Molecular Cloning: A Laboratory Manual", 1982).
  • the culture is centrifuged (5000 x g) to pellet the cells.
  • the cell pellet from a 330 mL E. coli culture is resuspended in 15 mL of sonication buffer (10 mM 2-amino-2-(hydroxymethyl) 1,3-propanediol hydrochloride (Tris-HCl), pH 8.0 + 1 mM ethylenediaminetetraacetic acid (EDTA).
  • sonication buffer 10 mM 2-amino-2-(hydroxymethyl) 1,3-propanediol hydrochloride (Tris-HCl), pH 8.0 + 1 mM ethylenediaminetetraacetic acid (EDTA).
  • resuspended cells are sonicated using the microtip probe of a Sonicator Cell Disruptor (Model W-375, Heat Systems-Ultrasonics, Inc., Farmingdale, New York). Three rounds of sonication in sonication buffer followed by centrifugation are employed to disrupt the cells and wash the inclusion bodies (IB). The first round of sonication is a 3 minute burst followed by a 1 minute burst, and the final two rounds of sonication are for 1 minute each.
  • Sonicator Cell Disruptor Model W-375, Heat Systems-Ultrasonics, Inc., Farmingdale, New York.
  • Three rounds of sonication in sonication buffer followed by centrifugation are employed to disrupt the cells and wash the inclusion bodies (IB). The first round of sonication is a 3 minute burst followed by a 1 minute burst, and the final two rounds of sonication are for 1 minute each.
  • the folded proteins can be affinity -purified using affinity reagents such as monoclonal antibodies or receptor subunits attached to a suitable matrix. Purification can also be accomplished using any of a variety of chromatographic methods such as: ion exchange, gel filtration or hydrophobic chromatography or reversed phase HPLC. These and other protein purification methods are described in detail (Methods in Enzymology, Volume 182 "Guide to Protein Purification” edited by Murray Deutscher, Academic Press, San Diego, California, 1990).
  • the purified protein is analyzed by RP-HPLC, electrospray mass spectrometry, and SDS-PAGE.
  • the protein quantitation is done by amino acid composition, RP-HPLC, and Bradford protein determination. In some cases tryptic peptide mapping is performed in conjunction with electrospray mass spectrometry to confirm the identity of the protein.
  • the mouse lymphoid cell line Baf3 was transfected with human granulocyte colony stimulating factor receptor (hG-CSFR) cDNA. Stable clones of Baf3 which expressed the G-CSFR and proliferated in the presence of hG-CSF were isolated and used to investigate the activity of human G-CSF receptor agonists without the influence of other human cytokine receptor responses.
  • hG-CSFR granulocyte colony stimulating factor receptor
  • the cDNA encoding hG-CSFR (a gift from Dr. Daniel C. Link (Washington University, St. Louis, MO) was released from the plasmid pEMCV.Sralpha as a .HmdIII/EcoRI (5' to 3') fragment, gel-purified, and inserted into the mammalian cell expression plasmid pcDNA3 (Invitrogen, San Diego, CA).
  • This plasmid contains enhancer-promoter sequences from the immediate early gene of the human cytomegalovirus (CMV), a bovine growth hormone polyadenylation signal and transcription termination sequences, a neomycin resistance gene is present for the selection of G418 stable cell clones, and an ampicillin resistance gene for selection in E. coli.
  • Ligation mixtures were transformed into E. coli strain TGI [delta (lac-pro), supE, thi, hsd ⁇ 5fF'(traO36, proA ⁇ * , lacF, Z ⁇ cZdeltaMl ⁇ ] and plasmid DNA was purified using a Qiagen Midiprep Plasmid Kit.
  • the structure of plasmid DNAs containing hG-CSFR were confirmed by restriction enzyme analysis and by automated DNA sequence analysis using an ABI sequencing machine. One of several plasmids with the correct structure was selected and given the designation pMON30298.
  • Ba ⁇ ceils maintained in complete growth medium (RPMI 1640 supplemented to 10% FBS and 10% Wehi 3B supernatant as a source for mouse IL- 3), were seeded at a subconfluent cell density of 10 ⁇ 5 cells/ml in growth media (RPMI 1640 5% FBS; 2 mM L-glutamine) the day prior to the electroporation.
  • the cells were collected and rinsed twice in 10 ml serum-free RPMI 1640.
  • the cells were diluted to 10 ⁇ 6/ml in serum-free RPMI and 1 ml was placed into each electroporation chamber (Gibco/BRL #1608AJ).
  • plasmid DNA 50 ug was added to each chamber and the chamber were incubated on ice for 30 minutes prior to electroporation.
  • the cells were electroporated on ice at a capacitance of 800 uF, - 400V, fast charge, and low ohms in a BRL CellPorator.
  • the cells were immediately removed from the chambers and placed into 10 cm dishes containing 10 ml of growth medium. The cells were allowed to recover for 48 hr in growth media prior to selection.
  • the cells were pelleted at 1000 rpm for 10 minutes, and resuspended into 10 ml of selection medium (growth medium containing 800 ug/ml G418 sulfate (Gibco/BRL).
  • selection medium growth medium containing 800 ug/ml G418 sulfate (Gibco/BRL).
  • the cells were kept in selection media, being passaged twice weekly, until only a few viable cells could be seen in the mock transfected control cell dishes (approximately 2 weeks).
  • the cells which had been electroporated with the hG-CSFR cDNA had grown to a cell density which allowed them to be tested for proliferation in the presence of hG-CSF (Fukunaga, R. et al., EMBO J. 10 (10): 2855-2865, 1991).
  • the cell proliferation assay conditions are as follows: Briefly, 25,000 cells were plated in a microtiter 96 well plate with or without cytokine in IMDM medium supplemented with BSA (50 ug/ml), human transferrin (100 ug/ml), lipid (50 ug/ml) 2-mercaptoethanol (50 uM final concentration). Each well was incubated with 0.5 uCi of 3 H-thymidine (16 hours) and the incorporated radioactivity was measured. Triplicate wells containing Baf3 cells were set up with 4 nM hG-CSF, 4 nM mIL-3 or media only control. Samples of different permuted proteins were tested in each assay.
  • Example 1 Construction of a permutein library without a linker region
  • Figure 1 shows a schematic of scanning permutagenesis.
  • a plasmid construct comprising a tandem repeat of the modified human granulocyte colony stimulating factor (hG-CSF with a serine for amino acid 17) gene joined by a sequence (GCCGG, termed a zero order linker) was generated and subcloned into the plasmid pACYC177 (Chang, A.C.Y. and S.N. Cohen, J Bacteriol. 134: 1141- 1156, 1978) using standard molecular biology methods (Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 2 nd edition, Cold Spring Harbor Press, New York, 1989).
  • the resultant plasmid construct (pMON15978) was linearized by restriction digestion (Smal) and used as a template for PCR amplification of circularly permuted hG-CSF (cphG-CSF) genes, following the method of Horlich (Horlick, R.A. et al., Protein Engineering 5: 427-431, 1992.
  • cphG-CSF circularly permuted hG-CSF
  • Figure 2 shows the position of the new amino termini for each new cphG-CSF.
  • cphG-CSF genes were inserted into phagemid presentation vector pCANTAB 5E (Pharmacia Biotech,) such that they were expressed as a part of a MPO species (Feng, Y., N. R. Staten, C. M. Baum, N. L. Summers, M. Caparon, S. C. Bauer, L. Zurfluh, J. P. McKearn, B. K. Klein, S. C. Lee, C. A. McWherter. 1997. Multi-functional hematopoeitic receptor agonists. World Patent Application WO 97/12985) which was in turn fused to the amino end of the phage genelll product.
  • the presented fusion protein contained, starting from its amino terminus, a hIL-3 receptor agonist, cphG-CSF, and the phage gene III product.
  • the juncture between the presented protein and the gene III product was as previously described (Merlin, S. et al., Applied Biochemistry and Biotechnology 67: 15-29, 1997).
  • phagemid particles were produced for each individual cphG-CSF-presenting species (Merlin et al., 1997). Some of these lots of particles were used to individually define the affinity properties of specific presented cphG-CSF species in analytical biopanning experiments (Caparon, M. H. et al., Molecular Diversity 1: 241-246, 1996; Merlin, S. et al., Applied Biochemistry and Biotechnology 67: 15-29, 1997), but all of the phage particle lots were titered and equivalent numbers of transducing units of each particle preparation were pooled together to form the scanning permutagenesis library for hG-CSF in an MPO background.
  • Figure 2 shows the MPO: cp hG-CSF species present in the library.
  • MPO cphG-CSF 38/37 is an example of the nomenclature used to specify the identity of individual permuted proteins. It describes a MPO molecule containing a circularly permuted human G-CSF module (with the serine 17 substitution). The first amino acid of the cphG-CSF domain is amino acid 38 of the parent protein, and the last amino acid is residue 37 of the parent.
  • Example 2 Presentation and Affinity screening of the MPO: cphGCSF library
  • MPO cphG-CSF 38/37
  • cphG-CSF 38/37 is a full hG-CSF receptor agonist (McKearn, J.P., Myelorestorative activities of synthokine and myelopoietin. In Proceedings of the 1996 IBC Conference on Therapeutic Applications of Cytokines, pp. 4.3.1-4.3.18, 1996). It was presented on filamentous phage as a positive control to demonstrate that permuted proteins can be presented on the surface of phage particles and - affinity selected. After phagemid particles were produced from this construct, they were subjected to analytical biopanning using cells expressing the hG-CSF receptor as affinity reagent.
  • Table 1 shows that phage presented MPO: cphG-CSF 38/37 was affinity selected by cells expressing the hG-CSF.
  • MPO: cphGCSF 38/37-GPIII fusion was expressed, secreted and assembled into phagemid particles, and could be affinity selected by the hG-CSF receptor. Permutagenesis of a protein does not appear to impair its successful presentation.
  • cp libraries Relative to typical phage display libraries, the complexities of cp libraries are low, containing perhaps hundreds to thousands of individuals.
  • the demonstration library here contained about 50 distinct clones, as opposed to more typical phage libraries containing more than 10 5 individuals (reviewed in Clackson, T. and J.A. Wells, Tibtech 12: 173-184, 1994).
  • MPO MPO-cphGCSF species
  • Five selectants had termini within helical domains of hG-CSF (MPO: cphG-CSFs 13/12, 19/18, 71/70, 123/122 and 159/158). For three of these molecules (MPO: cphG-CSFs 13/12, 71/70 and
  • Example 3 Biological activity of MPO: cphG-CSFs selected from the cp phage library
  • DNA segments containing individual affinity-selected MPO: cphGCSFs were subcloned into a mammalian expression vector, and expressed transiently in BHK cells as described above.
  • MPO cphG-CSFs isolated from biopanning were all expressed transiently in mammalian cells and the amount of MPO: cphG-CSF in each supernatant was determined by sandwich hIl-3 ELISA (Olins P.O. et al., J. Biol.
  • DH5 ⁇ TM F p/u80 dZ ⁇ cZdeltaM15, Life Technologies, Rockville, delta(Z ⁇ cZYA- ⁇ rgF)U169, c eoR, Maryland recAl, endAl, hsdRll (rk ,mk * ), phoA, supE44, lambda-, thi-1, gyrA96, relAl
  • JM101 delta (pro lac), supE, thi, Yanisch-Perron et al., Gene, 33:
  • pMON 15978 Amp R Plasmid construct comprising a This work tandem repeat of the modified human granulocyte colony stimulating factor (hG-CSF with a serine for amino acid 17) gene joined by a sequence (GCCGG, termed a zero order linker), subcloned into the plasmid pACYC177
  • pCANTAB 5E jjj pR Phage display vector containing Pharmacia lac promoter operably linked to Biotech, fd gene 3 signal sequence, a Piscataway, linker region, an E tag, and an NJ fd gene 3 structural gene all cloned into the vector backbone of pUC119 containing ColEl ori, the beta lactamase resistance gene, and an M13 ori.
  • PMON16016 A jj R Phagemid presentation vector This work pCANTAB ⁇ E derivation containing inserted individual cphG-CSF gene such that it was expressed as a part of an MPO species, fused in turn to the amino terminus end of the phage genelll product.
  • the first amino acid of the cphGCSF domain is amino acid 1 of the parent, and the last amino acid is residue 174 of the parent.
  • the zero order linker is attached at the carboxyl end of amino acid 174.
  • pMON16017 ⁇ j pR Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 3 of the parent, and the last amino acid is residue 2 of the parent.
  • pMON 16029 Amp R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 7 of the parent, and the last amino acid is residue 6 of the parent.
  • pMON16030 j npR Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 9 of the parent, and the last amino acid is residue 8 of the parent.
  • pMON16018 ⁇ R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 11 of the parent, and the last amino acid is residue 10 of the parent.
  • pMON16019 Amp R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 13 of the parent, and the last amino acid is residue 12 of the parent.
  • pMONl6020 A j npR Identical to pMONl6016 except This work the first amino acid of the cphG-CSF domain is amino acid 19 of the parent, and the last amino acid is residue 18 of the parent.
  • pMON16032 Amp R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 22 of the parent, and the last amino acid is residue 21 of the parent.
  • pMON 16033 Amp R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 27 of the parent, and the last amino acid is residue 26 of the parent.
  • pMON 16034 Amp R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino last amino acid is residue 30 of the parent.
  • pMON 16036 AmpR Identical to pMONl6016 except This work the first amino acid of the cphG-CSF domain is amino acid 37 of the parent, and the last amino acid is residue 36 of the parent.
  • pMON16037 Amp R Identical to pMONl6016 except This work the first amino acid of the cphG-CSF domain is amino acid 38 of the parent, and the last amino acid is residue 37 of the parent.
  • pMON 16038 Amp R Identical to pMONl6016 except This work the first amino acid of the cphG-CSF domain is amino acid 39 of the parent, and the last amino acid is residue 38 of the parent.
  • pMON 16039 j npR Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 43 of the parent, and the last amino acid is residue 42 of the parent.
  • pMON16040 AmpR Identical to pMONl6016 except This work the first amino acid of the cphG-CSF domain is amino acid 4 ⁇ of the parent, and the last amino acid is residue 44 of the parent.
  • pMON16041 Amp R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 47 of the parent, and the last amino acid is residue 46 of the parent.
  • pMON16022 Amp R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 49 of the parent, and the last amino acid is residue 48 of the parent.
  • pMON16042 Amp R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid ⁇ l of the parent, and the last amino acid is residue ⁇ O of the parent.
  • pMON 16043 Amp R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid ⁇ 3 of the parent, and the last amino acid is residue ⁇ 2 of the parent.
  • pMON 16044 Amp R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 56 of the parent, and the last amino acid is residue 5 ⁇ of the parent.
  • pMON 16023 AmpR Identical to pMONl6016 except This work the first amino acid of the cphG-CSF domain is amino acid 60 of the parent, and the last amino acid is residue ⁇ 9 of the parent.
  • pMON16024 Amp R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 67 of the parent, and the last amino acid is residue 66 of the parent.
  • pMON16047 AmpR Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 73 of the parent, and the last amino acid is residue 72 of the parent.
  • pMON16048 Amp Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 84 of the parent, and the last amino acid is residue 83 of the parent.
  • pMON16049 Amp R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 98 of the parent, and the last amino acid is residue 97 of the parent.
  • pMON160 ⁇ O Amp R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 100 of the parent, and the last amino acid is residue 99 of the parent.
  • pMON160 ⁇ l Amp R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 102 of the parent, and the last amino acid is residue 101 of the parent.
  • pMON160 ⁇ 2 AmpR Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 112 of the parent, and the last amino acid is residue 111 of the parent.
  • pMON160 ⁇ 3 mp Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 121 of the parent, and the last amino acid is residue 120 of the parent.
  • pMON16026 Amp R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 123 of the parent, and the last amino acid is residue 122 of the parent.
  • P MON16027 Amp R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 12 ⁇ of the parent, and the last amino acid is residue 124 of the parent.
  • pMON160 ⁇ 4 A p Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 133 of the parent, and the last amino acid is residue 132 of the parent.
  • pMON16055 AmpR Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 142 of the parent, and the last amino acid is residue 141 of the parent.
  • pMON160 ⁇ 6 Amp R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 143 of the parent, and the last amino acid is residue 142 of the parent.
  • pMON 16028 Amp R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 159 of the parent, and the last amino acid is residue 158 of the parent.
  • pMON16058 Amp Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 168 of the parent, and the last amino acid is residue 167 of the parent.
  • pMON 16059 Amp R Identical to pMON16016 except This work the first amino acid of the cphG-CSF domain is amino acid 170 of the parent, and the last amino acid is residue 169 of the parent.
  • Analytical biopanning shows that MPO molecules containing permuted hG- CSF domains can be presented and affinity selected in a hG-CSF receptor dependent fashion.
  • a mixture of phagemids presenting MPO: cphG-CSF 38/37 (ampicillin resistant) and M13k07 (kanamycin resistant) were exposed to BHK cells with or without the hG-CSF receptor on their surface, washed and eluted from the cell surface. Eluted phage were introduced into E. coli and the transfected cells were plated on media containing kanamycin or ampicillin. The ratio of ampicillin resistant to kanamycin resistant particles were determined prior to and following exposure to receptor by counting resistant colonies.
  • PCC_4G TATATAT GCGGCCGC GAA GGC CGG CAT GGC AGG CTG

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention porte sur un procédé de production de protéines à permutation circulaire (permutéines) réalisé par permutagenèse d'exploration. Ce procédé consiste à produire et introduire une série de gènes à permutation circulaire dans un vecteur d'affichage, exprimer ces gènes de sorte que les produits géniques soient localisés à la surface du vecteur d'affichage, générer une bibliothèque de vecteurs d'affichage présentant la protéine permutée, sélectionner par affinité les vecteurs d'affichage avec une protéine cible qui peut se lier à la protéine permutée, isoler et analyser des clones des vecteurs d'affichage sélectionnés de façon à identifier la protéine à permutation circulaire. L'invention porte également sur des procédés d'expression des permutéines et sur leurs utilisations.
EP99949630A 1998-09-25 1999-09-24 Procede de production de permuteines par permutagenese d'exploration Ceased EP1115854A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US10190898P 1998-09-25 1998-09-25
US101908P 1998-09-25
PCT/US1999/020891 WO2000018905A1 (fr) 1998-09-25 1999-09-24 Procede de production de permuteines par permutagenese d'exploration

Publications (1)

Publication Number Publication Date
EP1115854A1 true EP1115854A1 (fr) 2001-07-18

Family

ID=22287118

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99949630A Ceased EP1115854A1 (fr) 1998-09-25 1999-09-24 Procede de production de permuteines par permutagenese d'exploration

Country Status (3)

Country Link
EP (1) EP1115854A1 (fr)
AU (1) AU6246599A (fr)
WO (1) WO2000018905A1 (fr)

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5581476A (en) 1993-01-28 1996-12-03 Amgen Inc. Computer-based methods and articles of manufacture for preparing G-CSF analogs
CA2386090A1 (fr) * 1999-10-27 2001-05-03 California Institute Of Technology Production de genes et de proteines hybrides fonctionnels
EP2199393B1 (fr) 2000-04-17 2012-10-31 Dyax Corp. Nouveaux procédés de construction de bibliothèques de paquets génétiques qui affichent collectivement une famille diverse de peptides, polypeptides ou protéines
US8288322B2 (en) 2000-04-17 2012-10-16 Dyax Corp. Methods of constructing libraries comprising displayed and/or expressed members of a diverse family of peptides, polypeptides or proteins and the novel libraries
JP4860098B2 (ja) 2000-12-18 2012-01-25 ダイアックス、コープ 遺伝的パッケージの焦点を合わせたライブラリー
PL371781A1 (en) 2001-07-11 2005-06-27 Maxygen Holdings, Ltd. G-csf conjugates
US20040067532A1 (en) 2002-08-12 2004-04-08 Genetastix Corporation High throughput generation and affinity maturation of humanized antibody
GB0315182D0 (en) * 2003-06-28 2003-08-06 Asterion Ltd Cytokine variant polypeptides
US7381805B2 (en) 2005-06-01 2008-06-03 Maxygen Holdings, Ltd. Compositions comprising mixtures of positional PEG isomers of PEGylated G-CSF
EP3753947A1 (fr) 2007-09-14 2020-12-23 Adimab, LLC Bibliothèques d'anticorps synthétiques conçus de façon rationnelle et leurs utilisations
US8877688B2 (en) 2007-09-14 2014-11-04 Adimab, Llc Rationally designed, synthetic antibody libraries and uses therefor
US9873957B2 (en) 2008-03-13 2018-01-23 Dyax Corp. Libraries of genetic packages comprising novel HC CDR3 designs
CA2968164C (fr) 2008-04-24 2019-08-20 Dyax Corp. Bibliotheques de materiels genetiques comprenant de nouvelles conceptions cdr1, cdr2 et cdr3 hc et de nouvelles conceptions cdr1, cdr2 et cdr3 lc
MX360336B (es) 2010-07-16 2018-10-30 Adimab Llc Star Colecciones de anticuerpos.

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU717733B2 (en) * 1995-10-05 2000-03-30 G.D. Searle & Co. Novel G-CSF receptor agonists

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0018905A1 *

Also Published As

Publication number Publication date
WO2000018905A1 (fr) 2000-04-06
AU6246599A (en) 2000-04-17
WO2000018905A9 (fr) 2000-10-26

Similar Documents

Publication Publication Date Title
EP1660528B1 (fr) Muteines de lipocaline lacrymale
WO2000018905A1 (fr) Procede de production de permuteines par permutagenese d'exploration
US7691970B2 (en) Muteins of a bilin-binding protein with affinity for a given target
US7625700B2 (en) In vivo library-versus-library selection of optimized protein-protein interactions
CA2346122C (fr) Peptides bioactifs stabilises, procedes d'identification, synthese et utilisation
CA2381458A1 (fr) Procede de construction d'une d'adnc, nouveau polypeptide et adn codant ce polypeptide
WO2006040129A2 (fr) Conjugues de proteine utilisables en therapie, pour le diagnostic et en chromatographie
WO1995004069A1 (fr) Biotinylation de proteines
US20170305988A1 (en) Method of using a peptide library
EP1198586B1 (fr) Selection in vivo bibliotheque contre bibliotheque d'interactions proteine-proteine optimisees
JPH07503600A (ja) 改変された結合性を有する変異タンパク質の豊富化法
CA2549884A1 (fr) Domaines de liaison a la cytokine
JP2002506828A (ja) エリトロポエチン受容体のためのペプチドリガンド
WO1993012246A1 (fr) Vecteurs d'expression-secretion servant a produire des fragments fv biologiquement actifs
AU2004299160A1 (en) Cytokine binding domains

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20010321

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

17Q First examination report despatched

Effective date: 20021030

APBN Date of receipt of notice of appeal recorded

Free format text: ORIGINAL CODE: EPIDOSNNOA2E

APBR Date of receipt of statement of grounds of appeal recorded

Free format text: ORIGINAL CODE: EPIDOSNNOA3E

APAZ Date of receipt of statement of grounds of appeal deleted

Free format text: ORIGINAL CODE: EPIDOSDNOA3E

APAF Appeal reference modified

Free format text: ORIGINAL CODE: EPIDOSCREFNE

APAF Appeal reference modified

Free format text: ORIGINAL CODE: EPIDOSCREFNE

APBT Appeal procedure closed

Free format text: ORIGINAL CODE: EPIDOSNNOA9E

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED

18R Application refused

Effective date: 20060129