WO2000039310A9 - Proteines hybrides de rubredoxine, systeme et methodes d'expression de proteine - Google Patents

Proteines hybrides de rubredoxine, systeme et methodes d'expression de proteine

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Publication number
WO2000039310A9
WO2000039310A9 PCT/US1999/031176 US9931176W WO0039310A9 WO 2000039310 A9 WO2000039310 A9 WO 2000039310A9 US 9931176 W US9931176 W US 9931176W WO 0039310 A9 WO0039310 A9 WO 0039310A9
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WIPO (PCT)
Prior art keywords
rubredoxin
fusion protein
terminal
constituent
fused polypeptide
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Application number
PCT/US1999/031176
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English (en)
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WO2000039310A1 (fr
Inventor
Alan Przybyla
Nanda Menon
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Univ Georgia Res Found
Alan Przybyla
Nanda Menon
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Publication date
Application filed by Univ Georgia Res Found, Alan Przybyla, Nanda Menon filed Critical Univ Georgia Res Found
Priority to AU24869/00A priority Critical patent/AU2486900A/en
Publication of WO2000039310A1 publication Critical patent/WO2000039310A1/fr
Publication of WO2000039310A9 publication Critical patent/WO2000039310A9/fr

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    • 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
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • 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
    • C07K14/575—Hormones
    • C07K14/5759—Products of obesity genes, e.g. leptin, obese (OB), tub, fat
    • 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
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to a fusion protein comprising a fusion partner, in this case rubredoxin, fused directly or indirectly to a protein or peptide of interest, together with methods and materials for producing the fusion protein in a host cell and purifying the fusion protein.
  • the fusion protein can, in some embodiments of the invention, be cleaved to release the peptide or protein of interest for further use or analysis.
  • the invention further relates to immunogenic compounds comprising a rubredoxin as a carrier molecule linked to an antigen or a hapten.
  • Some of the strategies employed to overcome the problems of protein stability and solubility in E. coli include the use of fusion partners such as maltose binding protein (31 kD) (P. Riggs, in Ausebel, F.M. et al. (Eds)
  • fusion partners maltose binding protein, glutathione-S-transferase and thioredoxin are typically derived from bacteria or protozoa
  • the existence of closely related mammalian and avian analogues of these fusion partners makes them unsuitable for use as anchor proteins for haptens in antibody production or in vaccines.
  • continued development of new protein expression systems based on recombinant protein fusions with a stable carrier is necessary to advance the art of recombinant protein production.
  • the present invention provides a recombinant rubredoxin fusion protein containing an N-terminal rubredoxin constituent and a C-terminal fused polypeptide.
  • the fusion protein is capable of binding Fe 2+ when properly folded, giving it a red color that makes it easy to follow during purification.
  • the N- terminal rubredoxin constituent of the rubredoxin fusion protein preferably contains a rubredoxin obtained from an anaerobic bacterium, more preferably Desulfovibrio vulgaris, or a biologically active analogue, fragment, or modification thereof.
  • the C-terminal fused polypeptide can be a polypeptide that is insoluble or known to form inclusion bodies in a host cell.
  • amyloid peptide, leptin, proinsulin, trypsin inhibitor, and the extracellular domain of luteinizing hormone receptor can be fused to rubredoxin to yield rubredoxin fusion proteins of the invention.
  • the linkage between the N-terminal rubredoxin constituent and C-terminal fused polypeptide can, but need not, be a cleavable linkage.
  • Antigenic or immunogenic rubredoxin fusion proteins of the invention have C-terminal fused polypeptides that are antigens (including polyfusion antigens) or haptens.
  • the rubredoxin constituent serves as the carrier molecule to yield an immunogenic fusion product. Because rubredoxin itself is only negligibly antigenic, there is no need to include in the antigenic or immunogenic fusion protein a cleavage site to allow cleavage of the N-terminal rubredoxin constituent from C-terminal fused polypeptide.
  • the invention includes a method for producing an antibody to a C-terminal fused polypeptide by eliciting in a host cell, preferably a mammalian host cell, an immune response to a rubredoxin fusion protein containing the C-terminal fused polypeptide.
  • the antibodies thus generated can be polyclonal or monoclonal, and are preferably not, but can be, cross-reactive with rubredoxin.
  • the invention further provides a polypeptide vaccine containing an antigenic or immunogenic rubredoxin fusion protein of the invention, and a polynucleotide vaccine containing a polynucleotide encoding an antigenic or immunogenic rubredoxin fusion protein.
  • the N-terminal rubredoxin constituent of the rubredoxin fusion protein can be directly or indirectly linked to the C-terminal fused polypeptide.
  • the fusion protein contains a spacer region positioned between the N-terminal rubredoxin constituent and the C-terminal fused polypeptide. This intervening spacer region optionally contains a proteolytic cleavage site, an affinity purification sequence, or both.
  • the N-terminal rubredoxin constituent can be directly linked to the C-te ⁇ ninal fused polypeptide, with no intervening spacer region.
  • the present invention further provides a recombinant polynucleotide having a nucleotide sequence that encodes a rubredoxin fusion protein as described herein.
  • the invention includes an expression vector that contains a promoter operably linked to a nucleotide sequence encoding a rubredoxin fusion protein, and a host cell transformed with an expression vector comprising a recombinant polynucleotide comprising a nucleotide sequence encoding a rubredoxin fusion protein.
  • the host cell is a bacterial cell.
  • an expression vector that contains a nucleotide sequence encoding rubredoxin or a biologically active analogue, fragment, or modification thereof; an intervening nucleotide sequence encoding a spacer region; and a multiple cloning region that contains at least one restriction endonuclease recognition site.
  • the intervening nucleotide sequence preferably includes all or a portion of the multiple cloning region, and the spacer region encoded by the intervening nucleotide sequence preferably contains at least one of one of a proteolytic cleavage site and an affinity purification sequence.
  • a preferred expression vector is pRUBEX3.
  • the invention further provides a method for making a rubredoxin fusion protein that involves introducing into a host cell a recombinant polynucleotide having a nucleotide sequence encoding a rubredoxin fusion protein, followed by expressing the fusion protein in the host cell.
  • the fusion protein is removed from the host cell and further purified as desired.
  • the fusion protein contains an affinity purification sequence that permits reversible binding of the fusion protein to an affinity chromatography matrix thereby facilitating removal of contaminants.
  • the invention also provides a recombinant method for making a polypeptide that includes introducing into a host cell a recombinant polynucleotide having a nucleotide sequence encoding a rubredoxin fusion protein; expressing the fusion protein in the host cell; removing the fusion protein from the host cell; and cleaving the fusion protein to yield the rubredoxin constituent and the polypeptide.
  • this method further includes separating the polypeptide from the rubredoxin constituent after cleavage.
  • Figure 1 depicts (a) a schematic of the vector pRUBEX3, including the Multiple Cloning Region (MCR); and (b) the nucleotide sequence (SEQ ID NO : 1 ) of a portion of pRUBEX3 together with the amino acid sequence encoded thereby (SEQ ID NO:2) wherein the 52 amino acids of rubredoxin (SEQ ID NO:3) are underlined; the amino acids of the polyhistidine (polyHis) sequence (i.e., His-His-His-His-His-His) (SEQ ID NO:4) are in bold; the eight amino acids of the flag peptide are double-underlined (DYKDDDDK; i.e., Asp- Tyr-Lys-Asp-Asp-Asp-Asp-Lys) (SEQ ID NO:5); the five amino acids of the enterokinase site (DDDDK; i.e., Asp-Asp-Asp-A
  • pRUBEX3 includes, in place of the polyhistidine sequence, the affinity tag His-Gly-Leu-His (SEQ ID NO: 7).
  • Figure 2 shows a portion of the nucleotide sequence (SEQ ID NO: 7
  • the underlined amino acid sequence (SEQ ID NO: 10) represents the A ⁇ ,_ 42 peptide and the intervening spacer region comprises a flag peptide sequence (SEQ ID NO:5), a polyhistidine (polyHis) sequence for use in affinity purification (SEQ ID NO:4), and the amino acid sequence IEGR (in bold) (i.e., Ile-Glu-Gly-Arg) (SEQ ID NO:l 1). which is the recognition site for the restriction protease Factor Xa.
  • Another embodiment of the A ⁇ M2 rubredoxin fusion construct includes, in place of the polyhistidine sequence, the affinity tag His-Gly-Leu-His (SEQ ID NO:7).
  • Figure 3 is a schematic of the expression vector pRUBEX2-LHR, which contains the amino-terminal 298 amino acid residues of human luteinizing hormone receptor (LHR), representing the extracellular domain, cloned into the Ndel/BamRl site of pRUBEX2; the resulting construct encodes a fusion protein consisting of rubredoxin followed by a spacer region comprising a polyhistidine tag to facilitate purification of the fusion protein and a Factor Xa recognition site that directly precedes the LHR coding region.
  • Another embodiment of pRUBEX2-LHR includes, in place of the polyhistidine sequence, the affinity tag His-Gly-Leu-His (SEQ ID NO:7).
  • Figure 4 is a schematic of the expression vector pRUBEXl-LHR, which contains cDNA encoding the amino-terminal 340 amino acids of human luteinizing hormone receptor (LHR), representing the extracellular domain, cloned into the .B ⁇ mHI site of pRUBEXl ; the resulting construct encodes a fusion protein consisting of the N-terminal extracellular domain of human LHR directly fused to the carrier protein rubredoxin at the C-terminal end of rubredoxin.
  • LHR luteinizing hormone receptor
  • Figure 5 shows Tris-tricine gel electrophoresis of rubredoxin fusion proteins and digestion products.
  • Figure 6 is a Western-blot analysis of purified pig leptin/rubredoxin fusion protein and a Factor Xa digest of the fusion protein.
  • Rubredoxin is an electron carrier protein originally isolated and then cloned from the anaerobic sulfate reducing bacteria, Desulfovibrio vulgaris. Since then, rubredoxins from several different anaerobic organisms have been discovered and characterized. Rubredoxin is a small redox protein (5.6 kD) carrying a single non-haem iron center. The crystal structure of the protein has been solved and reveals a free carboxy-terminal end, making it well-suited for fusing peptides. The iron center imparts a red color to the protein (absorption maxima at 390 nm and 495 nm) providing a visible marker for easy monitoring during purification protocols.
  • the red color also serves indicator as to whether the fusion protein has folded correctly, since an incorrectly folded protein will not bind the metal.
  • Recombinant rubredoxin can be produced at high levels (50- 60 mg/L of purified protein) in E. coli and is very soluble, biologically active and stable. Conveniently, rubredoxin is a thermostable protein and can withstand 70°C-80°C for more than an hour without denaturation. It also retains its metal center in denaturing agents like 0.5% SDS and 6M urea.
  • the present invention utilizes rubredoxin as a protein fusion partner in the creation of a simple, reliable, reproducible, scalable and economical recombinant protein expression system.
  • the presence of a correctly folded fusion protein can be visually tracked during purification due to the effects of the iron atom.
  • proper folding of the fused protein may be facilitated by the redox functionality of rubredoxin. That is, the presence of high levels of an active, foreign electron carrier protein like rubredoxin is likely to beneficially alter the redox microenvironment of the fusion protein. Folding of a protein fused to an electron carrier protein such as rubredoxin is thus likely to be affected by the redox state of the carrier as well as the oxidation state within the cell.
  • the protein expression system of the invention is particularly useful for producing proteins and peptides, such as ⁇ -amyloid peptide, leptin and pro- insulin, that are otherwise insoluble or tend to form inclusion bodies in recombinant systems.
  • proteins and peptides such as ⁇ -amyloid peptide, leptin and pro- insulin
  • leptin from both rat and pig are known to form inclusion bodies and require the use of chaotropic agents for solubilization, and pig leptin can be efficiently produced using the protein expression system of the present invention.
  • the invention provides a rubredoxin fusion protein and, further, a recombinant polynucleotide containing a nucleotide sequence that encodes the rubredoxin fusion protein of the invention, as well as a polynucleotide having a nucleotide sequence complementary thereto.
  • a rubredoxin fusion protein is a protein that comprises a rubredoxin constituent and a polypeptide of interest.
  • the rubredoxin constituent comprises the N- terminus of the fusion protein, and the fused polypeptide constitutes the C- terminus of the fusion protein.
  • the rubredoxin fusion protein contains an intervening spacer region between the rubredoxin constituent and the fused polypeptide, as described more fully below.
  • the rubredoxin constituent of the rubredoxin fusion protein is composed primarily of a rubredoxin polypeptide and serves as a "carrier" or
  • the rubredoxin constituent can assist in stabilization, folding, solublization and/or targeting of the fused polypeptide, while providing additional options for detecting, isolating and purifying the polypeptide.
  • the rubredoxin constituent of the rubredoxin fusion protein optionally contains one or more of an affinity purification sequence (described below), a signal sequence or a targeting sequence, for example a sequence targeting the fusion protein to a bacterial periplasm or causing the fusion protein to be secreted into the surrounding media, which is particularly useful in eukary otic expression systems.
  • a signal sequence or targeting sequence is preferably located at the N-terminus of the rubredoxin fusion protein (and hence is located at the N-terminal end of the rubredoxin constituent), whereas an affinity purification sequence can be positioned at the N-terminus of the fusion protein, within the rubredoxin polypeptide sequence itself, or C- terminal to the rubredoxin polypeptide.
  • the affinity purification sequence may be thought of as part of the intervening spacer region rather than part of the rubredoxin constituent per se. Inclusion of the optional affinity sequence, signal sequence and/or targeting sequence must not prevent the rubredoxin polypeptide from folding properly.
  • the rubredoxin polypeptide folds properly can be easily assayed by determining whether it can bind a divalent cation, particularly Fe 2+ , as discussed in more detail below.
  • a divalent cation particularly Fe 2+
  • engineering a histidine tag His-His-His-His-His-His-His, SEQ ID NO:4 as an affinity purification sequence at the N-terminus of the fusion protein caused the rubredoxin polypeptide to fail to bind iron.
  • use of an N-te ⁇ ninal affinity sequence that is less highly charged could result in a rubredoxin polypeptide that does bind iron. i.e.. a rubredoxin fusion protein of the invention.
  • the rubredoxin fusion protein is a single polypeptide chain wherein the rubredoxin constituent is linked by way of a peptide bond, either directly or indirectly, to the polypeptide of interest. This linkage is termed “direct” in embodiments of the rubredoxin fusion protein containing no intervening spacer region; it is termed “indirect” in embodiments of the rubredoxin fusion protein that contain an intervening spacer region.
  • the fused polypeptide can have a preselected or predetermined amino acid sequence, a random amino acid sequence, or an unknown amino acid sequence.
  • peptide polypeptide
  • protein as used herein are interchangeable, as the invention is not limited by the length or the function of the amino acid sequence linked to the rubredoxin constituent. As used herein these terms all refer generally to a plurality of amino acids joined together in a linear chain via peptide bonds.
  • peptide may be used to connote a shorter polypeptide such as dipeptide, tripeptide, or oligopeptide; the term oligopeptide typically connoting a polypeptide having between 2 and about 50 or more amino acids.
  • peptide is not limited to polypeptides of any particular length.
  • protein is sometimes used herein to mean a functionally folded polypeptide of any length having structural, enzymatic or other active properties. Regardless of the nomenclature used, however, no limitations on the length or the function of the fused polypeptide or protein are intended.
  • the rubredoxin constituent of the fusion protein comprises a rubredoxin polypeptide.
  • the rubredoxin polypeptide has the wild- type amino acid sequence of a rubredoxin protein obtained from an anaerobic bacterium, preferably from Desulfovibrio, Clostridium, Desulfoarculus or
  • Pyrococcus spp. more preferably from D. vulgaris, D. vulgaris (Hildenborough), C. pasteurianum, C. butyricum, D. baarsii or P. furiosa.
  • GenBank Accession numbers for nucleotide sequences encoding rubredoxins include D76419 (rub gene for D. vulgaris), M28848 (rub gene for D. vulgaris (Hildenborough), M60116 (C. pasteurianum rubredoxin gene), YI 1875 (C. butyricum rubredoxin gene), and X99543 for D. baarsii.
  • a particularly preferred amino acid sequence for the rubredoxin polypeptide is an amino acid sequence of a rubredoxin from D.
  • the amino acid sequence of the rubredoxin polypeptide useful in the fusion protein of the invention is not intended to be limited to the exact wild-type amino acid sequence of naturally occurring rubredoxin proteins; rather, the rubredoxin polypeptide includes biologically active analogues, fragments, or modifications of any and all naturally occurring rubredoxin proteins.
  • biologically active means that the rubredoxin analogue, fragment or modification thereof can, when present as a component of the fusion protein of the invention, can bind a divalent cation.
  • biologically active rubredoxin or analogue, fragment, or modification thereof binds Zn 2+ or Fe 2+ ; more preferably it binds Fe 2+ .
  • Biological activity e.g., iron- binding activity
  • iron binding can be visually detected because the bound complex is red. Binding of Fe 2+ by the fusion protein is indicative of proper folding of its rubredoxin polypeptide.
  • rubredoxin is a small protein; for example, rubredoxin from D. vulgaris contains about 52 amino acids.
  • a "fragment" of rubredoxin means a rubredoxin that has been truncated at the C-terminus; preferably, the fragment is at least about 40 amino acids in length, more preferably it is at least about 45 amino acids in length.
  • an "analogue" of rubredoxin means a rubredoxin that contains one or more amino acid substitutions, deletions, additions, or rearrangements.
  • an amino acid belonging to a grouping of amino acids having a particular size or characteristic such as charge, hydrophobicity and hydrophilicity
  • a rubredoxin polypeptide useful in a fusion protein according to the invention includes a rubredoxin that contains amino acid substitutions at sites such that the iron-binding activity of the polypeptide is not eliminated.
  • Substitutes for an amino acid may be selected from other members of the class to which the amino acid belongs.
  • nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine.
  • Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • a rubredoxin analogue has at least about 80% amino acid identity with a reference rubredoxin protein; more preferably it has at least about 90% amino acid identity with a reference rubredoxin protein.
  • the reference rubredoxin protein is preferably a rubredoxin from D. vulgaris; more preferably it is SEQ ID NO:3.
  • Amino acid identity is defined in the context of a homology comparison between the rubredoxin analogue and the reference rubredoxin protein.
  • the two amino acid sequences are aligned in a way that maximizes the number of amino acids that they have in common along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to maximize the number of shared amino acids, although the amino acids in each sequence must nonetheless remain in their proper order.
  • the percentage amino acid identity is the higher of the following two numbers: (a) the number of amino acids that the two polypeptides have in common within the alignment, divided by the number of amino acids in the rubredoxin analogue, multiplied by 100; or (b) the number of amino acids that the two polypeptides have in common within the alignment, divided by the number of amino acids in the reference rubredoxin protein, e.g., SEQ ID NO:3, multiplied by 100.
  • Modified rubredoxin includes rubredoxins chemically or enzymatically derivatized at one or more constituent amino acid, including side chain modifications, backbone modifications, and N- and C- terminal modifications including acetylation, hydroxylation, methylation, amidation, and the attachment of carbohydrate or lipid moieties, cofactors, and the like.
  • the fused polypeptide of the rubredoxin fusion protein can be a polypeptide that has, in the past, been difficult to isolate in biologically active form using other recombinant expression systems.
  • polypeptides include, for example, hydrophobic peptides, (that is, peptides that are insoluble in aqueous solutions), peptides and proteins that produce insoluble sedimentation aggregates known as "inclusion bodies" when overexpressed (e.g., amyloid peptides, such as ⁇ -amyloid 1-42 peptide and ⁇ -amyloid 1-40 peptide, leptins, including pig leptin and rat leptin, preproinsulin, trypsin inhibitor, and the extracellular domain of luteinizing hormone receptor), and those that become insoluble when present the high concentrations found in typical protein overproduction systems.
  • amyloid peptides such as ⁇ -amyloid 1-42 peptide and ⁇ -amyloid 1-40 peptide
  • the rubredoxin fusion protein of the invention in contrast, is preferably soluble in aqueous solutions. More preferably, the rubredoxin fusion protein does not form insoluble sedimentation aggregates during recombinant overproduction of the fusion protein; that is, it remains soluble when overexpressed in the host cell. "Overexpression" in this context means expression of the rubredoxin fusion protein at a level of at least about 10 mg fusion protein per 100 mL cell extract (i.e., about 100 mg/L).
  • aggregates of the rubredoxin fusion protein do form, they are preferably capable of being resolubilized using a nonionic detergent to yield a fusion protein having a biologically active (i.e., iron-binding) rubredoxin constituent.
  • a biologically active i.e., iron-binding
  • the rubredoxin fusion protein of the invention when it binds Fe 2+ , is detectably labeled as a result of its red color.
  • the rubredoxin fusion protein is further detectably labeled.
  • the detectable label is a radioisotope, a heavy isotope, or a fluorescent label.
  • Isotope labels can be conveniently incorporated into the fusion protein using isotopically labeled amino acids or precursor compounds during synthesis in the host cell using methods well known in the art. Examples of useful radiolabels include 3 H, 14 C and 35 S; useful heavy isotope labels are exemplified by 13 C and 15 N.
  • a preferred fluorescent label is isofluorothiocyanate (IFTC), which can be chemically attached to the fusion protein following biosynthesis.
  • a particularly preferred embodiment of the fusion protein of the invention comprises a rubredoxin constituent fused, directly or indirectly, to an amyloid peptide.
  • the amyloid peptide is ⁇ -amyloid 1-40 or ⁇ -amyloid 1-42, or a biologically active analogue, modification or derivative thereof.
  • Amyloid peptides that are isotopically labeled, as described above, are also especially useful.
  • a biologically active ⁇ -amyloid peptide is one that retains the ability to aggregate into fibrils such as are observed in Alzheimer's plaques.
  • tyrosine at the 10 position in ⁇ -amyloid can be changed to tryptophan to yield a bioactive ⁇ -amyloid peptide analogue, and the tryptophan can be detectably labeled using IFTC to generate modified bioactive peptide having a chartreuse color.
  • IFTC ⁇ -amyloid peptide analogue
  • the production of biologically inactive amyloid fusion proteins for instance those having one or two amino acid deletions, additions or changes that reduce or eliminate aggregation activity, is useful for comparative or mechanistic studies and is also encompassed by the present invention.
  • arginine at the 5 position in ⁇ -amyloid can be changed to cysteine to yield a ⁇ -amyloid peptide analogue, and the cysteine can be labeled with IFTC to generate modified amyloid peptide that is less biologically active than the naturally occurring peptide.
  • fusion protein of the invention is a fusion protein comprising a rubredoxin constituent linked, directly or indirectly, to the extracellular domain of luteinizing hormone receptor (LHR) or biologically active fragment, modification or analogue thereof.
  • LHR luteinizing hormone receptor
  • Another embodiment of the invention that is particularly well suited for use in generating mammalian antibodies to the fused polypeptide is a rubredoxin fusion protein comprising an N-terminal rubredoxin constituent directly linked to a C-terminal fused polypeptide antigen or hapten.
  • a hapten is a low-molecular weight compound that reacts specifically with an antibody but does not stimulate antibody production (i.e., is not antigenic) unless complexed with a carrier protein.
  • the carrier protein i.e., rubredoxin
  • the hapten portion of the immunogenic rubredoxin fusion protein is preferably at least about four amino acids in length, more preferably at least about six amino acids in length, most preferably at least about eight amino acids in length, and is preferably less than about 50 amino acids in length, more preferably less than about 35 amino acids in length, most preferably less than about 25 amino acids in length.
  • polypeptide antigen that is advantageously linked to the rubredoxin constituent in this embodiment of the rubredoxin fusion protein is a protein that would be insoluble or form inclusion bodies in the absence of a rubredoxin carrier.
  • the polypeptide antigen portion of the rubredoxin fusion protein can contain more than one antigenic epitope fused in tandem, forming what is known as a polyfusion antigen.
  • Rubredoxin has a significant advantage over other known carrier proteins for antibody production (such as thioredoxin, glutathione sulfotransferase and maltose binding protein) in that rubredoxins are never present in mammalian systems.
  • the corresponding recombinant polynucleotide encoding this embodiment of the rubredoxin fusion protein includes, in the 5' to 3' direction, a nucleotide sequence encoding the rubredoxin constituent directly followed by an in-frame nucleotide sequence encoding the fused polypeptide.
  • a rubredoxin fusion protein comprising a fused polypeptide antigen can, if desired, contain one or both of a cleavage site between the rubredoxin polypeptide and the fused polypeptide antigen, and an affinity purification sequence.
  • a preferred embodiment of the invention includes a rubredoxin fusion protein comprising a rubredoxin constituent that is linked indirectly to the fused polypeptide.
  • the rubredoxin fusion protein comprises an intervening spacer region positioned between the rubredoxin constituent and the fused polypeptide. The invention is not to be limited by any particular upper limit on the size of the spacer region.
  • the optimal length of the spacer region depends on the nature of the fused peptide and can be readily determined by one of skill in the art. For example, where the spacer region contains a cleavage site, the optimal length of the spacer region can be determined by analyzing the efficiency of cleavage in test fusion proteins having spacer regions of varying lengths. Preferably, the intervening spacer region consists of less than about 100 amino acids.
  • the spacer region preferably contains between 0 and about 100 amino acids, more preferably between about 10 and about 60 amino acids, more preferably between about 20 and about 40 amino acids.
  • the spacer region preferably contains between 0 and about 100 amino acids, more preferably between about 10 and about 60 amino acids, more preferably between about 20 and about 40 amino acids.
  • the intervening space region (MHGGSEFENHHHHHHNDYKDDDDKDLIEGR (i.e., Met-His-Gly-Gly-Ser- Glu-Phe-Glu-Asn-His-His-His-His-His-His-His-His-His-Asn-Asp-Tyr-Lys-Asp-Asp-Asp- Asp-Lys-Asp-Leu-Ile-Glu-Gly-Arg.
  • SEQ ID NO:12 for the rubredoxin/ ⁇ - amyloid fusion protein consists of 30 amino acids.
  • An analogous intervening spacer region that includes a His-Gly-Leu-His (SEQ ID NO: 7) affinity tag contains 28 amino acids.
  • the intervening spacer region optionally comprises one or more proteolytic cleavage sites, one or more affinity purification sequences, and/or one or more amino acids that happen to be encoded by that portion of the multiple cloning region of the vector positioned between the nucleotide sequence encoding the rubredoxin constituent and nucleotide sequence encoding the fused polypeptide, as described in more detail below.
  • the proteolytic cleavage site allows enzymatic or chemical cleavage of the fusion protein into two portions, permitting separation of the fused polypeptide from the rubredoxin constituent. Thus, it must be positioned in between the rubredoxin constituent and the fused polypeptide to serve its intended function. Preferably, it is positioned at the end of the intervening spacer region so as to minimize the attachment of additional amino acids to the fused polypeptide.
  • Chemical cleavage can be achieved, for example, by cyanogen bromide or hydroxylamine.
  • a cleavage site that comprises methionine allows cleavage to release the polypeptide of interest upon contact of the rubredoxin fusion protein with cyanogen bromide.
  • Enzymatic cleavage can be facilitated by including as a cleavage site an amino acid sequence recognized by a restriction protease, also called an endoprotease.
  • a restriction protease also called an endoprotease.
  • cleavage sites recognized by thrombin, Factor Xa, renin, or enterokinase can be utilized.
  • cleavage of the rubredoxin fusion protein at the cleavage site yields a polypeptide having no extraneous. unintended or non-native N-terminal amino acids.
  • An affinity purification sequence is an amino acid sequence designed to facilitate purification of the fusion peptide using affinity chromatography.
  • a polyhistidine (SEQ ID NO:4) or His-Gly-Leu- His (SEQ ID NO:7) site or "tag” can be engineered into the fusion protein to allow purification of the fusion protein using Ni-chelating affinity chromatography (commercially available from numerous sources, for example Qiagen, Boehringer Mannheim Biochemicals, and Novagen).
  • an affinity purification system commercially available from IBI Kodak (Rochester, NY) utilizes the "flag" peptide (YKDDDDK, i.e., Tyr-Lys- Asp-Asp- Asp- Asp-Lys, SEQ ID NO: 13) and a monoclonal antibody-linked resin (IGM2) that is highly specific for that peptide.
  • YKDDDDK i.e., Tyr-Lys- Asp-Asp- Asp- Asp-Lys, SEQ ID NO: 13
  • IGM2 monoclonal antibody-linked resin
  • a chitin-binding tag can be combined with a self-cleaving protein splicing element (an intein) to permit purification of the rubredoxin fusion protein and cleavage of the fused polypeptide in a single chromatographic step.
  • an intein self-cleaving protein splicing element
  • the fusion protein binds to a chitin column.
  • a disulfide reducing agent such as dithiothreitol, ⁇ -mercaptoethanol or cysteine
  • the intein undergoes specific self-cleavage which releases the fused polypeptide from the chitin-bound intein tag.
  • an affinity purification sequence can be positioned at essentially any location along the length of the rubredoxin fusion protein as long as it does not prevent the rubredoxin polypeptide from folding properly.
  • the recombinant polynucleotide of the invention includes a nucleotide sequence encoding the rubredoxin fusion protein of any of the various embodiments described above.
  • the recombinant polynucleotide encodes, in a 5' to 3' direction, a rubredoxin constituent linked, directly or indirectly, to a polypeptide of interest; alternatively it encodes, in the 5' to 3' direction, a polypeptide of interest linked, directly or indirectly, to a rubredoxin constituent. It optionally encodes an intervening spacer region, one or more affinity sites, cleavage sites, targeting sites, and the like, as described generally for the rubredoxin fusion protein.
  • the invention further provides an expression vector capable of directing expression of a rubredoxin fusion protein in a host cell.
  • the expression vector can be circular or linear, single-stranded or double stranded, and can include DNA, RNA, or any modification or combination thereof.
  • the vector can be a plasmid, a viral vector or a cosmid. Selection of a vector or plasmid backbone depends upon a variety of desired characteristics in the resulting construct, such as a selection marker, plasmid reproduction rate, and the like. Suitable plasmids for expression in E.
  • coli for example, include pUC(X), pKK223-3, pKK233-2, pTrc99A, and pET-(X) wherein (X) denotes a vector family in which numerous constructs are available.
  • pUC(X) vectors can be obtained from Pharmacia Biotech (Piscataway, NH) or Sigma Chemical Co. (St. Louis, MO).
  • pKK223-3, pKK233-2 and pTrc99A can be obtained from Pharmacia Biotech.
  • pET-(X) vectors can be obtained from Promega (Madison, WI) Stratagene (La Jolla, CA) and Novagen (Madison, WI).
  • the vector preferably includes an origin of replication (known as an "orf ) or replicon.
  • an origin of replication known as an "orf ) or replicon.
  • ColEl and P15A replicons are commonly used in plasmids that are to be propagated in E. coli.
  • the expression vector preferably takes the form of a DNA molecule containing a nucleotide sequence encoding the rubredoxin fusion protein of the invention, and optionally includes a promoter sequence operably linked to the coding sequence.
  • a promoter is a DNA fragment that facilitates transcription of genetic material. Transcription is the formation of an RNA chain in accordance with the genetic information contained in the DNA.
  • the invention is not limited by the use of any particular promoter, and a wide variety are known.
  • Promoters act as regulatory signals that bind RNA polymerase in a cell to initiate transcription of a downstream (3' direction) coding sequence.
  • a promoter is "operably linked" to a nucleotide sequence if it does, or can be used to, control or regulate transcription of that nucleotide sequence.
  • the promoter used in the invention can be a constitutive or an inducible promoter. It can be, but need not be, heterologous with respect to the host cell.
  • Preferred promoters for bacterial transformation include lac, / ⁇ cUV5, tac, trc, T7, SP6 and ara.
  • the expression vector optionally includes a Shine Dalgarno site (e.g., a ribosome binding site), and a start site (e.g., the codon ATG) to initiate translation of the transcribed message to produce the enzyme. It can also include a termination sequence to end translation. A termination sequence is typically a codon for which there exists no corresponding aminoacetyl-tRNA, thus ending polypeptide synthesis.
  • the expression vector optionally further includes a transcription termination sequence.
  • the rrnB terminators which is a stretch of DNA that contains two terminators, Tl and T2, is the most commonly used terminator that is incorporated into bacterial expression systems (J. Brosius et al., J. Mol Biol, 148:107-127 (1981)).
  • the expression vector optionally includes one or more marker sequences, which typically encode a gene product, usually an enzyme, that inactivates or otherwise detects or is detected by a compound in the growth medium.
  • a marker sequence can render the transformed cell resistant to an antibiotic, or it can confer compound-specific metabolism on the transformed cell.
  • Examples of a marker sequence are sequences that confer resistance to kanamycin, ampicillin, chloramphenicol and tetracycline.
  • the expression vector comprises a nucleotide sequence encoding a rubredoxin polypeptide and a multiple cloning region for the insertion of a polypeptide of interest.
  • the multiple cloning region comprises at least one restriction site and preferably comprises a multiplicity of restriction sites (see, for Example, Fig. 1 showing the multiple cloning region of pRUBEX3).
  • the multiple cloning region (sometimes referred to as a polyclonal site) is positioned such that cloning a nucleotide sequence encoding a polypeptide of interest into that site will permit expression of a rubredoxin fusion protein comprising the polypeptide of interest; for example, the polypeptide of interest will be in frame with respect to the rubredoxin constituent and the intervening spacer region, if it is present.
  • the expression vector comprises a nucleotide sequence encoding rubredoxin or a biologically active analogue, fragment, or modification thereof, an intervening nucleotide sequence, and a multiple cloning region comprising a multiplicity of restriction endonuclease recognition site.
  • the intervening nucleotide sequence preferably encodes at least one of a proteolytic cleavage site and an affinity purification sequence.
  • expression vectors include pRUBEXl, in which the coding sequence for D. vulgaris rubredoxin and the fused polypeptide are directly linked; i.e., there is no intervening spacer region between the two components; pRUBEX2, which contains an intervening spacer region comprising a histidine tag and a Factor Xa cleavage site; and pRUBEX3, which, in addition to the histidine tag and a Factor Xa cleavage site of pRUBEX2, contains as part of the intervening spacer a portion of a multiple cloning region to facilitate cloning of the nucleotide sequence encoding the fused polypeptide into the vector.
  • the invention also provides a method for making a rubredoxin fusion protein.
  • An expression vector as described above that contains a nucleotide sequence capable of directing expression of a rubredoxin fusion protein is introduced into a host cell and the rubredoxin fusion protein is then expressed in the transformed cell.
  • Any suitable host cell can be used, without limitation.
  • the expression vector is a DNA molecule that comprises a nucleotide sequence encoding the rubredoxin fusion protein.
  • the expression vector comprises RNA
  • the host cell preferably comprises a reverse transcriptase enzyme in order to facilitate expression of the rubredoxin fusion protein.
  • Viral vectors are especially useful in eukaryotic protein expression systems, which facilitate protein glycosylation.
  • the fusion protein can be removed from the transformed host cell and purified.
  • the rubredoxin fusion protein can be labeled with a radioisotope such as 3 H, 13 C, i5 N or 35 S during synthesis using methods well-known in the art.
  • the host cell in which the rubredoxin fusion protein is expressed in accordance with the present invention can be a bacterium, a protozoan, or a eukaryotic cell.
  • Eukaryotic cells include, for example, plant cells and animal cells, including for example mammalian cells, yeast cells and insect cells.
  • the fusion protein is preferably targeted to the endoplasmic recticulum.
  • Suitable host cells can be differentiated or undifferentiated, and include cells growing in mammalian tissue culture, including hybridoma cells. Particularly suitable host cells are those that have been used in other protein expression systems, such as E. coli, Bacillus spp., and Streptomyces spp. Methods of introducing expression vectors into host cells are well-known in the art; electroporation is preferred.
  • Rubredoxin fusion proteins that contain a polyhistidine (S ⁇ Q ID NO:4) or His-Gly-Leu-His (S ⁇ Q ID NO:7) tag can be purified by Ni-chelating chromatography.
  • Imidazole can be used to elute the fusion protein.
  • purification can be achieved at moderate temperatures using a single affinity chromatographic step.
  • Ni-chelating chromatography can be performed at temperatures from about 4°C to about 60 °C, depending on the thermal stability of the fused polypeptide; typically the process is performed at room temperature or colder temperatures.
  • the affinity chromatography can be followed with high performance liquid chromatography for further purification of the fusion proteins.
  • the invention further provides a method for making a polypeptide using the protein expression system described herein.
  • a rubredoxin fusion protein comprising a cleavage site is expressed in a host cell as described herein, then removed from the host cell.
  • the rubredoxin fusion protein can be affinity purified at this point, if it also contains an affinity purification sequence.
  • the polypeptide of interest is then chemically or enzymatically cleaved away from the rubredoxin constituent of the fusion protein.
  • a preferred cleavage site comprises Ile-Glu-Gly-Arg (I ⁇ GR, S ⁇ Q ID NO: 11) and the restriction protease Factor Xa is used to cleave the fusion protein to obtain the free polypeptide.
  • the free polypeptide can be further purified away from the rubredoxin constituent by reverse phase chromatography, typically at about pH 6 to about pH 8.5, depending on the stability of the polypeptide to acid and base.
  • reverse phase chromatography is preferably carried out at temperatures between about 45 °C and about 65 °C, although reverse phase high pressure liquid chromatography for most other polypeptides is typically carried out at room temperature or colder temperatures.
  • Other useful restriction proteases include thrombin. renin, and enterokinase, provided their recognition site has been engineered into the intervening spacer region of the fusion protein.
  • Cyanogen bromide can also be used if a methionine intervenes between the peptide of interest and the rubredoxin component, provided the peptide of interest contains no internal methionines that would result in undesired cleavage of the peptide upon contact with CNBr.
  • the invention further provides a method for making antibodies to a polypeptide of interest (i.e., a polypeptide antigen or hapten) using a rubredoxin fusion protein.
  • a rubredoxin fusion protein comprising a rubredoxin polypeptide and the polypeptide antigen or hapten is introduced into a host, eliciting an immune response to the peptide antigen in a host cell.
  • a cleavage site between the rubredoxin component and the fused polypeptide is not required as the rubredoxin moiety is negligibly antigenic.
  • the fusion protem used in this method preferably does not contain a cleavage site.
  • the method for making antibodies is not limited by the selection of a particular host; rather any desired host can be used such as a rabbit, goat, mouse, rat, cow or chicken.
  • Antibodies are isolated and purified from the host using methods well-known in the art.
  • the antibody is preferably a polyclonal antibody; however, the rubredoxin fusion protein can also be used to generate monoclonal antibodies to the polypeptide of interest.
  • the invention also provides a polypeptide vaccine comprising a rubredoxin fusion protein of the invention and a polynucleotide vaccine comprising a polynucleotide comprising a nucleotide sequence encoding a rubredoxin fusion protein.
  • a preferred rubredoxin fusion protein for use in this embodiment of the invention includes, for example, a rubredoxin constituent linked to a polypeptide antigen or hapten.
  • the rubredoxin fusion protein used in or encoded by the vaccine is one wherein the N-terminal rubredoxin constituent is directly linked to the C-terminal fused polypeptide.
  • a vaccine is capable of generating an immune response in the animal to which it is administered.
  • An immune response includes either or both of a cellular immune response or production of antibodies, and can include activation of the subject's B cells, T cells, helper T cells or other cells of the subject's immune system.
  • Immunogenicity of rubredoxin fusion protein can be determined, for example, by administering the adjuvanted fusion protein to the subject, then observing of the associated immune response by analyzing antibody titers in the subject's serum.
  • the rubredoxin fusion protein used in the vaccine or encoded by the polynucleotide used in the vaccine further includes at least one epitope or epitope mimic, such as a T cell, helper T cell or B cell epitope or epitope mimic.
  • Epitopes or epitope mimics can be derived from the species to which the vaccine is to be administered, from the species that was the source of the polypeptide antigen or hapten, or from any other species, including a virus, bacterium, or parasite.
  • a polynucleotide encoding a rubredoxin fusion protein can include DNA, RNA, a modified nucleic acid, or any combination thereof.
  • the polynucleotide can be supplied as part of a vector or as a "naked" polynucleotide.
  • Polynucleotide vaccines are known in the art, e.g. F. Vogel et al., Clin. Microbiol. Rev. 8:406-410 (1995).
  • Polynucleotides can be generated by means standard in the art, such as by recombinant techniques, or by enzymatic or chemical synthesis.
  • a polynucleotide used in a vaccine of the invention is preferably one that functionally encodes a rubredoxin fusion protein.
  • a protein is "functionally encoded” if it is capable of being expressed from the genetic construct that contains it.
  • the polynucleotide can include one or more expression control sequences, such as cz ' s-acting transcription/translation regulatory sequences, including one or more of the following: a promoter, response element, an initiator sequence, an enhancer, a ribosome binding site, an RNA splice site, an intron element, a polyadenylation site, and a transcriptional terminator sequence, which are operably linked to the coding sequence and are, either alone or in combination, capable of directing expression in the target animal.
  • expression control sequences such as cz ' s-acting transcription/translation regulatory sequences, including one or more of the following: a promoter, response element, an initiator sequence, an enhancer, a ribosome
  • An expression control sequence is "operably linked" to a coding sequence if it is positioned on the construct such that it does, or can be used to, control or regulate transcription or translation of that coding sequence.
  • Preferred expression control sequences include strong and/or inducible cis-acting transcription/translation regulatory sequences such as those derived from metallothionine genes, actin genes, myosin genes, immunoglobulin genes, cytomegalovirus (CMN), SV40, Rous sarcoma virus, adenovirus, bovine papilloma virus, and the like.
  • the coding and expression control sequences for the rubredoxin fusion protein are preferably constructed in a vector, such as a plasmid of bacterial origin, a cosmid, episome, or a viral vector, for administration to a target animal.
  • a vector useful in the vaccine of the present invention can be circular or linear, single-stranded or double stranded.
  • a specific embodiment employs constructs using the plasmid pcD ⁇ A3.1 as the vector (InVitrogen Corporation, Carlsbad, CA).
  • the vector construct can contain immunostimulatory sequences (ISS) that stimulate the animal's immune system.
  • ISS immunostimulatory sequences
  • nucleotide sequences coding cytokines such as granulocyte macrophage colony stimulating factor (GM-CSF) or interleukin-12 (IL-12).
  • cytokines can be used in various combinations to fine-tune the response of the animal's immune system, including both antibody and cytotoxic T lymphocyte responses, to bring out the specific level of response needed to affect the animal's reproductive system.
  • the vector can be a viral vector, including an adenovirus vector, and adenovirus associated vector, or a retro viral vector.
  • the viral vector is a nonreplicating retroviral vector such as the Moloney murine leukemia virus (N2) backbone as described by Irwin et al. (J. Virology 68:5036-5044 (1994)).
  • the polypeptide or polynucleotide vaccine is admimstered in a manner and an amount effective to cause the desired immune response in the animal.
  • a polypeptide vaccine can be administered in one or more doses, and typically includes between about 10 ⁇ g to about 2 mg of rubredoxin fusion protein.
  • a polynucleotide vaccine containing polynucleotide in an amount of about 5 ⁇ g to about 500 ⁇ g can be administered in one or more doses.
  • One of skill in the art can readily determine a suitable dosage for a particular animal, depending on the nature, size and overall health of the animal, as well as the condition to be treated.
  • a polypeptide or polynucleotide vaccine of the invention can be administered in any convenient manner.
  • forms of administration include intramuscular administration, subcutaneous or intradermal administration, oral administration, as by food or water, topical administration, including transdermal administration, aerosol administration, cloacal or vaginal administration, intracoelomic administration, intranasal administration, and transconjunctival administration, including the use of eye drops.
  • liposome-mediated, microsphere-mediated, and microencapsulation systems are all included as delivery vehicles for the vaccine of the present invention.
  • the vaccine includes an adjuvant, the selection of which is a matter well-known to those of skill in the art and is influenced by the nature of the intended recipient.
  • the pET-24a expression system utilizes the bacteriophage T7 promoter that serves as a binding site for T7 RNA polymerase and was incorporated into the chromosomal DNA of E. coli strain BL21 (DE3) (Novagen).
  • T7 RNA polymerase is synthesized only upon the addition of isopropyl ⁇ -D- thiogalactoside (IPTG) to growing cultures since the gene for the T7 polymerase has been spliced into the chromosomal DNA of the E. coli host.
  • IPTG isopropyl ⁇ -D- thiogalactoside
  • the pET-24a plasmid also contains the gene for kanamycin resistance for selection of plasmid- containing colonies.
  • a nucleotide sequence encoding the flag peptide (YKDDDDK, SEQ ID NO: 13) affinity tag (IBI/KODAK, Rochester, NY), a polyhistidine sequence, and an enterokinase protease site was attached in frame at the C-terminal end of the rubredoxin gene, yielding pRUBEXl.
  • the encoded peptide sequence provides two independent sites for affinity purification of the fusion protein along with a protease site for removal of the protein of interest from the fusion.
  • a resulting fusion protein can be purified by Ni-chelating affinity chromatography due to the presence of the polyhistidine tag, and the flag peptide offers a second method for affinity purification using the monoclonal antibody-linked resin (IGM2) available from IBI Kodak.
  • IGM2 monoclonal antibody-linked resin
  • All plasmids containing fusion constructs were transformed into E. coli strain BL-21, and the host cells were grown induced as described above for the rubredoxin optimization. After induction, the temperature was brought to 20 °C for the final 7 hour growth period. Cells were harvested and stored at - 70 °C until needed. For expression of 15 N-labeled proteins and peptides, cultures were grown in M9 minimal media. Cells were initially streaked on M9 minimal media plates containing 50ug/ml kanamycin. A well-isolated colony was transferred to 100ml of M9 minimal media containing lg/L ammonium- 15 N chloride. The culture was grown at 37°C overnight.
  • additional FeSO 4 was added to bring the final concentration to 80uM.
  • the cultures were induced with ImM IPTG at an OD 590 of 1 and were then transferred to 20°C and allowed to grow for an additional 15 hours. Cells were harvested and stored at - 70°C until needed.
  • Frozen cell paste (12-15 grams, representing cells from 3 liters of media) was suspended in 100ml phosphate buffer (20mM, pH 7.4; 0.5M NaCl; Buffer A) and the resuspended cells were sonicated using a Branson Ultrasonic disrupter for 15 minutes (10 second pulses). The cell sonicate was spun at
  • High flow metal-chelating columns (5ml; Pharmacia) were used for purification of the fusion proteins.
  • the column was washed and charged with 0.1 M NiSO 4 , washed again and then equilibrated with Buffer A containing 25mM imidazole. Imidazole was added to the cell-free extract to give a final concentration of 25 mM.
  • This material was loaded onto the column at 3ml min and was washed with the equilibration buffer until the flow through was clear (4- 6 bed volumes).
  • the column was subsequently washed with 4 bed volumes of Buffer A containing 75mM and 150mM imidazole in order to elute several incomplete fusion products which were most likely formed as a result of incomplete translation.
  • the complete fusion protein was finally eluted with Buffer A containing 300mM imidazole. Elution of the fusion proteins was monitored during purification by visual inspection of the column and flow through since the fusion products are red in color (due to the iron-sulfur center of rubredoxin).
  • the purified protein (approximate volume 50-60 ml) was dialyzed overnight in 4 liter batches against a total of 12 liters of Tris HCl buffer (20mM, pH7.5). Total protein obtained after purification was estimated using the BCA assay (Pierce Biochemicals) with BSA as the standard.
  • the dialyzed fusion protein was brought to a concentration of 5-6 mgs/ml using an Amicon Centriprep (10K cut off) and was filtered using a 0.22 ⁇ m syringe filter (Whatman) prior to storage in sterile falcon tubes.
  • the protein keeps well at a concentration of 5-6mgs/ml at 4°C at pH 8.0 in the dark. Prolonged exposure to light (as in cold cabinets) leads to photobleaching of the protein and formation of a precipitate.
  • DAEFRHDSGYE VHHQKLVFFAEDVGSNKGA ⁇ GLMVGGVVPA], SEQ ID NOS: 10 and 14 generated by proteolytic cleavage of a membrane bound pre- protein (APP) represents a major constituent of the senile plaques which are deposited in the brains and cerebrovasculature of patients affected by Alzheimer's disease.
  • the plaques are formed by ordered, self-aggregation of the peptides to form amyloid fibers. Onset of this disease is marked by enhanced levels of the longer and more hydrophobic A ⁇ M2 peptide in the brain (Iwatsubo et al., Neuron 13:45-53 (1994)); Lemere et al, Nat. Med.
  • Labeling of peptides with the non-radioactive isotopes l3 N and 13 C greatly simplifies structural determination via NMR and would greatly benefit determination of structural changes that occur during the aggregation process, but chemical synthesis of such labeled peptide is prohibitively expensive. Labeled peptides and proteins are easy to produce using recombinant techniques and are much less costly than those produced synthetically making this method very attractive to groups pursuing structural data.
  • ⁇ -amyloid peptides 1-40 and 1-42 were synthesized as soluble recombinant fusion proteins using rubredoxin as a fusion partner.
  • the fusion protein was purified by Ni-chelating chromatography and average yields of purified fusion product varied from 40-50 mg/L of culture.
  • the fusion product was cleaved by restriction protease Factor Xa to separate the ⁇ -amyloid peptide from the rubredoxin carrier.
  • the peptide was further purified by reverse phase chromatography at pH 6-8.5 at temperatures between about 45-65 °C. The quality of the peptide was consistent from batch to batch and showed no chemical modification as judged by mass spectrometric analysis.
  • the purified peptides were biologically active and formed fibers at pH 2.5 as well as pH 6.5.
  • the DNA sequence encoding the ⁇ -amyloid 1-42 peptide was amplified by PCR using the human Alzheimers precursor protein (human ⁇ APP) gene as template (provided by Dr. Sangram Sisodia, Johns Hopkins University, Boston, MA). During the PCR process (Bej et al., Crit. Rev. Biochem. Mol Biol.
  • a restriction protease site for Factor Xa was introduced at the amino terminal end of the ⁇ -amyloid 1-42 peptide for proteolytic cleavage from rubredoxin, in that the N-terminus primer designed and used for amplifying the ⁇ -amyloid 1-42 sequence encoded the residues Ile-Glu-Gly-Arg, the tetrapeptide recognition site for Factor Xa, along with a Pstl restriction site (35 bases total).
  • the C-terminus primer contained the sequence for the C-terminal region of the relevant peptide followed by a Kpnl restriction site.
  • the amplified DNA product was digested by Pstl and Kpnl and was ligated into the Pstl-Kpnl site of the poly linker region of pRUBEX3 (Example I) and sequenced.
  • the final construct encoded a 13.6 kD fusion protein containing the rubredoxin gene, the His-Flag affinity site, the Factor Xa restriction site and the ⁇ -amyloid 1 -40 or 1 - 42 peptides (Fig. 2). All constructs were initially made in pUC 18, sequenced and then transferred into the expression vector pET24a at the Nde-BamHl site.
  • Expression of the fusion protein in one liter cultures was carried out essentially as described above in Example I.
  • Expression in 20L fermentors were started by inoculating 50mls of overnight culture into a 24L fermentors containing 20L of LB supplemented with lOOuM FeSO 4 .
  • the culture was grown at 37°C with stirring at 240 rpm and 3L of air/min.
  • At an OD of 1.2 IPTG was added to a final concentration of lmM and the temperature lowered to 20 °C.
  • Cultures were allowed to grow for another 6 hours. Cells were harvested and stored at -70°C. Average cell yield from a 20L fermentor run was 5.5-6g/liter. Cells were disrupted, and Ni-chelating chromatography was carried out, substantially as described in Example I.
  • Low temperature purification is of further advantage since the stability and possible biohazards of subjecting peptides incorporating S 35 methionine and the non-radioactive isotopes ⁇ 15 or C 13 to temperatures above 60 ° C are not known.
  • Load capacity of a semi-preparative column in this material (10mm x 25cm) with good resolution of peaks was in the range of 100- 200ugs of ⁇ -amyloid 1-42 peptide. It is expected that load levels in the range of 1.5-2mgs per run (25mm diameter X 25cm length) can be achieved. This would minimize loss in recovery of the peptide because of multiple runs and make the procedure much more economical.
  • a soluble rubredoxin ⁇ -amyloid fusion protein was produced.
  • the rubredoxin moiety folded correctly as judged by the successful incorporation of iron into the protein.
  • the fusion protein was easily purified by Ni-chelating chromatography.
  • Ni-chelating resins from several companies can be used (for example, Qiagen, Invitrogen and Boehringer Mannheim Biochemicals), but they do differ in binding and elution characteristics with respect to imidazole concentrations.
  • the red color of the fusion provided a visible intrinsic marker to follow the protein during purification.
  • Typical yields of the fusion protein were in the range of 40-50mgs/L as estimated by the BCA method.
  • the fusion protein remained soluble at concentrations of 5-6mgs/ml at 4°C.
  • the average yield of ⁇ -amyloid 1-40 or ⁇ -amyloid 1-42 peptide was 3-4mgs/L. These recoveries can be further improved by employing larger columns and reducing the number of chromatographies to purify 3-4mg of peptide from 20 to one. Additionally, one of the main problems with expressing eukaryotic proteins in bacterial hosts is the altered bias in codon usage. By altering the codons of the eukaryotic gene to coincide with bacterial usage (where feasible), it is probable that higher yields can be obtained. According to these data, decreasing the expression temperature may also lead to higher yields.
  • a major advantage of this recombinant system is the possibility of synthesizing radioactive peptides using S 35 -labeled methionine. Purification of this peptide is possible at moderate temperatures of 45-50°C, conditions under which S 35 is stable. Another advantage of this system is that it can be used for incorporating N 15 , C 13 and, with appropriate auxotrophs, various labeled amino acids into the ⁇ -amyloid peptides.
  • LHR luteinizing hormone receptor
  • the coding region of the D. vulgaris rubredoxin gene was cloned into the expression vector pET16b (Novagen), which contains a Factor Xa site at the appropriate location, at the Xbal/Ncol site to yield pRUBEX2.
  • LHR human luteinizing hormone receptor
  • a Factor Xa recognition site directly precedes the LHR coding region, and a spacer region is located between the rubredoxin coding region and the LHR coding region, thus including the Factor Xa site (Fig. 3).
  • the spacer region further contains a poly- histidine tag to facilitate purification of the fusion protein.
  • the total length of the spacer region 50 amino acids, which is longer than just the affinity sequence and the Factor Xa recognition sequence, was chosen to maximize the efficiency of Factor Xa cutting to insure efficient separation of rubredoxin and LHR fragments after isolation of the fusion construct.
  • the column was washed with four bed volumes of equilibration buffer and then four bed volumes of equilibration buffer containing 150mM imidazole.
  • the fusion protein was eluted with Buffer A containing 300mM imidazole. This process could be monitored by the intrinsic red color of the fusion protein.
  • the purified protein was dialyzed overnight against three 4L changes of 20mM Tris-HCl pH 7.5.
  • the fusion protein was then concentrated and washed with the Tris-HCl buffer using an Amicon Centriprep (10K exclusion) filter to remove all traces of imidazole, since imidazole is an inhibitor of Factor Xa protease. Digestion and cleavage of the rubredoxin-LHR fusion protein.
  • Fusion protein was digested with protease Factor Xa (Boehringer Mannheim) at a ratio of 250:1 (fusiomprotease) at 37°C for 45 minutes with constant stirring. This protocol resulted in the cleavage of about 95% of the fusion protein.
  • the Xa-digested material was adjusted to 25mM imidazole and passed once again over the metal-chelating Sepharose resin. In this instance, the rubredoxin, which retained the poly-Histidine on its carboxy-terminus, bound to the resin while the LHR fragment passed through the column.
  • the flow-through was successively dialyzed as follows: 1) for 3 hours in IL of 50mM Tris-HCl pH 7.5, 10% glycerol and ImM Cysteine; 2) for 3 hours in IL of 50mM Tris- HCl pH 7.5, 10% glycerol, ImM cysteine and ImM cystine; and 3) overnight in 2L of 50mM Tris-HCl pH 8.0, 5mM DTT and 10% glycerol. The dialyzed material was concentrated and used for further experiments.
  • the rubredoxin protein expression system produced 20-40mg/L of rubredoxin-LHR fusion protein.
  • the fusion protein could be purified to greater than 95% purity by passage over a single Ni-Sepharose column. Although a second passage produced greater purity, it did not result in a more homogeneous LHR preparation and gave lower yields as would be expected.
  • the fusion protein was readily cleaved by low concentrations of Factor Xa provided that the Ni-Sepharose eluate had been thoroughly dialyzed to remove all traces of imidazole. Repassage over the Ni-Sepharose column resulted in the binding of all of the rubredoxin (and the red coloration); the LHR moiety was, in contrast, included in the flow through from the column. This step removed over 95% of the rubredoxin fusion partner and this purity could be improved by a second passage over the column with relatively small losses.
  • the recombinant LHR fragment cross reacted with LHR antibodies and could be used as an antigen for the production of polyclonal antibodies.
  • the pRUBEX2 vector was not designed to produce a recombinant fusion protein that is secreted, and thus does not effect proper folding of some mammalian polypeptides that contain disulfide linkages.
  • the extracellular domain of LHR for example, contains at least four disulfide bonds; this apparently prevented it from folding to the native conformation in the reducing environment of the E. coli cytosol, a result which was not unexpected.
  • rubredoxin- LHR fusion that is targeted for secretion would be expected to fold properly in the more oxidized periplasmic environment where the dsb protein, which is involved in disulfide bond formation and shuffling in E. coli, is present.
  • the coding region of the D. vulgaris rubredoxin gene was cloned into the expression vector pET21b (Novagen) at the Ndel/BamHl site to yield pRUBEXl.
  • a cDNA encoding the amino-terminal 340 amino acids of human luteinizing hormone receptor (hLHR), representing the extracellular domain (see Example III) was then cloned into the Bam l site of pRUBEXl to yield pRUBEXl-LHR which thus encodes a fusion protein consisting of the N- terminal extracellular domain of human LHR fused to the carrier protein rubredoxin at the C-terminal end of rubredoxin (Fig. 4).
  • E. coli strain BL21 cells were transformed with pRUBEXl -LHR and a 1.0ml overnight culture of the transformed cells was inoculated into a 100ml culture and grown for 3 hours at room temperature prior to induction with ImM IPTG for 3 hours at room temperature.
  • Cells were collected at 5000 x g for 10 minutes and stored overnight at -20 °C and then resuspended in 10ml of 50mMTris-HCl, pH 7.5 and disrupted with 5 second bursts of a sonicator at full power until all cells were broken. The lysate was centrifuged at 25,000 x g for 15 minutes and the supernatant was discarded.
  • the pellet was washed successively in water, 50mM Tris-HCl ph 7.5 containing 5mM EDTA, 50mM Tris-HCl pH 7.5, containing 5mM EDTA and 0.4% Triton X-100, water, and 50mM Tris-HCl, pH 7.5, containing ImM EDTA.
  • the washed pellet was solubilized in 5.0ml of 8M urea, and EDTA and phenylmethylsulfonylfluoride (PMSF) were added to final concentrations of 5mM and 2mM respectively.
  • PMSF phenylmethylsulfonylfluoride
  • the solubilized protein was cleared for 30 minutes at 25,000 x g and then successively dialyzed as follows: 1) for 3 hours in 200ml of 50mM Tris-HCl, pH 7.5, 10%) glycerol, and ImM cysteine (Sigma Chemical Co., St. Louis, MO); 2) for 3 hours in IL of 50mM Tris-HCl, pH 7.5, 10% glycerol, ImM cysteine, and ImM cystine (Sigma Chemical Co., St. Louis, MO); and 3) overnight in IL of 50mM Tris-HCl pH 8.0, 5mM DTT, and 10% glycerol.
  • the dialyzed material was fractionated in SDS polyacrylamide gels and a 3 kD band which was specific to transformed cells and immunoreactive with human LHR antibodies, was cut from preparative gels and washed with water.
  • the excised bands were lyophilized, ground into powder and injected into rabbits. The initial injection was in Freund's complete adjuvant (Pierce Biochemicals) and was followed by three boosts in Freund's incomplete adjuvant (Pierce Biochemicals). Animals were bled and an IgG fraction was prepared from the serum.
  • Wild type rat LHR cDNA was cloned into pET24 to form pCDNA3.
  • pCDNA3 and the empty vector pET24, as a control, were transiently transfected with lipofectamine into monkey kidney (COS 7) cells grown in
  • DMEM Dulbecco's modified Eagle ' s medium; Gibco/BRL
  • 10% fetal bovine serum Gibco/BRL
  • the cells were chilled on ice, washed with phosphate buffered saline, and extracted in 150mM NaCl, 20mM HEPES pH 7.4 (Sigma Chemical Co., St. Louis, MO) and 0.5% Nonidet-P40 (Sigma Chemical Co., St. Louis, MO) in the presence of
  • the induced rubredoxin-LHR fusion protein was readily visible by Coomassie Blue staining after fractionation of whole bacterial cell lysates from transformed BL21 E. coli cells in SDS polyacrylamide gels. Large amounts of the fusion protein were produced; estimates from the stained gels suggest that from 10-20mg of fusion protein was produced in 500ml of cells. The fusion protein was easily centrifuged from cell lysates, but it was also readily soluble in 8M urea.
  • Fusion protein bands excised from SDS-polyacrylamide gels and ground into a fine powder were excellent antigens in New Zealand which rabbits. Although three boosts were administered before bleeding the animals, it is not known if they were all necessary.
  • the IgG fraction purified from the sera of inoculated rabbits did not react with native human LHR or rubredoxin, but reacted only with the recombinant hLHR fusion protein or deglycosylated native hLHR.
  • the fusion protein expressed in E. coli that was used for antigen is not glycosylated, it is not surprising that antisera directed against the fusion protein did not react with native hLHR which contains six known N-linked glycosylation sites.
  • Leptins are 12-15 kDa proteins which are known to be involved in the regulation ofobesity in humans and other mammalian organisms. Expression of various leptins (human, rat, mouse and pig) by themselves or as fusions in E. coli have invariably led to the formation of inclusion bodies (K. Giese et al., Mol Med. 2: 50-58 (1996); A. Fawzi et al., Horm. Metab. Res. 28: 694-697 (1996)). The inclusion bodies can be resolubilized and the proteins refolded to yield active leptin with varying degrees of success.
  • Native pig leptin contains a 21 amino acid signal peptide which is absent in the mature processed protein.
  • this signal peptide sequence was deleted so that the ammo-terminus originated at Val-22 of the pre-leptin sequence.
  • the N-terminus primer was designed according to the amyloid protein scheme and included the Kpnl restriction site and a Factor Xa recognition site just before the initial residues of the leptin sequence.
  • the C-terminus primer contained the sequence for the C- terminal region of the protein along with a Hindlll restriction site. The gene was synthesized by PCR amplification using a cDNA clone as the template.
  • the amplified product was digested with Kpnl and Hindlll and was ligated into the corresponding site of pRUBEX 3 (Example I). After transformation of the plasmid into E. coli DH5 ⁇ (strain BL-21 as described in Example I), three recombinant clones were isolated and determined by restriction analysis to contain the entire fusion protein gene.
  • the fusion protein was purified as described in Examples I and II, and the yield of the soluble leptin fusion was about 10-15mgs/liter.
  • Leptin fusions were digested with Factor Xa at a ratio (w/w) of 100: 1 at pH 8.0 at room temperature. Leptin fusions were also digestible with recombinant enterokinase, but not with native enterokinase. The digest was centrifuged at 15,000xg for 15 minutes and the supernatant was used for analysis.
  • the purified fusion protein and the Factor Xa digests were analyzed on a 10% Tris-tricine/sodium dodecyl sulfate (SDS) polyacrylamide gel (see Fig. 5).
  • Lane 4 shows purified, undigested fusion protein (arrow; 22 kD, 5 ⁇ g) but the mobility of the band is retarded due to the presence of the histidine moiety due to its positive charge; lanes 5 and 6 show a 7-hour digest of fusion protein (15 ⁇ g and lO ⁇ g, respectively) with Factor Xa.
  • the 14 kD band represents pure leptin and the 9.3 kD band (bottom arrow) represents the rubredoxin-histidine portion of the fusion just before the Factor Xa site. Again, the mobility of the 9.3 kD band is retarded due to the presence of the histidine moiety. The presence of leptin in the supernatant indicates that leptin is soluble after digestion with the protease. These results were confirmed by western-blot analysis (Fig. 6). Lane 1 contains pig leptin fusion protein digested with Factor Xa (150ng); Lane 2 contains purified pig leptin fusion protein (lOOng).
  • the membrane was exposed to fluorescent-labeled antibody raised against purified pig leptin.
  • the two lanes shown in Fig. 6 were cross-reacted to antibody raised against pig leptin. Both of the products cross-reacted with the antibody thereby indicating the presence of leptin in the fusion and in the digested fusion.
  • pro-insulin synthesized in E. coli is the major source of pharmaceutical grade insulin used in the treatment of diabetes.
  • insulin is initially produced as a pro-insulin chain composed of three domains, A, B, and C, which contain two intramolecular disulfide bonds.
  • domain C is cleaved from the A and B domains and the result is a heterodimeric insulin molecule whose two subunits are joined by two disulfide bonds.
  • two strategies have been employed for the synthesis of mature insulin.
  • One strategy involves reconstitution of the separately synthesized subunits, A and B, to form active insulin while the second strategy involves synthesizing the pro-insulin (all three domains) as an insoluble single chain in inclusion bodies.
  • subunit C After successful solubilization and refolding of the pro-insulin, subunit C is removed by cleavage with trypsin and carboxypeptidase C to yield active insulin.
  • the latter method has been reported to give significantly higher levels of active insulin, although pro-insulins from different animal sources have different intrinsic solubilities.
  • a feline pro- insulin/rubredoxin fusion was therefore constructed in order to more efficiently recover soluble fusion protein. Construction of the feline pro-insulin/rubredoxin fusion
  • the gene encoding feline pro-insulin was synthesized as constituent oligonucleotides which were ligated together to form a single composite gene.
  • the codons were altered according to an E. coli codon usage table to maximize expression.
  • a Factor Xa site was included at the 5' end of the pro-insulin oligonucleotide containing the N-terminus sequence.
  • the composite gene was then digested with Kpnl-Hindlll and was ligated into the corresponding sites of RUB ⁇ X 3 and finally transformed into E coli DH5 ⁇ (strain BL-21 as described in Example I).
  • E coli DH5 ⁇ strain BL-21 as described in Example I.
  • the fusion protein was purified as described in Examples I and II, and the yield of the soluble pro-insulin fusion was about 25 mgs/liter.
  • Pro- insulin fusions were digested with Factor Xa at a ratio (w/w) of 100:1 at pH 8.0 at room temperature.
  • Pro-insulin fusions were also digestible with recombinant enterokinase, but not with native enterokinase. Digestion with enterokinase reduced the amount of non-specific cleavage products compared to Factor Xa.
  • the rubredoxin pro-insulin fusion migrated as a 19 kD band on a 10% Tris-Tricine native gel ( Figure 7, lane 1, 5 ⁇ g).
  • Digests of the fusion with Factor Xa showed a number of non-specific cleavage products; therefore, digestion with recombinant enterokinase (an enterokinase site being a part of the flag peptide sequence) was attempted.
  • the fusion protein was digested at a w/w ratio of 75: 1 (fusiomenzyme) overnight at room temperature. The digest was centrifuged at 15,000 x g for 20 minutes and the supernatant was analyzed on a 10% Tris- Tricine gel.
  • the digest revealed the two expected bands: a 9.6kDa rubredoxin band (top arrow) and a 9 kD pro-insulin band (bottom arrow; Figure 7, lanes 2 and 3, lO ⁇ g and 7 ⁇ g, respectively).
  • the mobility of the 9.6 kD band was retarded due to the presence of the histidine moiety.
  • the 9 kD band (bottom arrow) was electrophoretically transferred to a PVDF membrane and was analyzed via amino acid sequencing. The first twenty amino acids were determined and were found to match the expected sequence of pro-insulin, except for an additional portion of the polylinker which was present as a result of the location of the enterokinase restriction site in the fusion protein.

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Abstract

Cette invention a trait à une nouvelle protéine hybride de recombinaison comprenant de la rubrédoxine comme partenaire de fusion. Cette protéine hybride peut, éventuellement, comprendre une région d'espacement intervenante entre le constituant rubrédoxine et le polypeptide fusionné étudié, pouvant comporter un site de clivage protéolytique pour la libération dudit polypeptide. Cette protéine hybride peut contenir un ou plusieurs sites pour purification par affinité. L'invention concerne également des méthodes et des substances permettant de produire et d'utiliser la protéine hybride rubrédoxine. Elle porte, en outre, sur des composés et des compositions antigéniques, notamment des vaccins, renfermant la rubrédoxine comme molécule porteuse liée à un antigène ou à un haptène.
PCT/US1999/031176 1998-12-29 1999-12-29 Proteines hybrides de rubredoxine, systeme et methodes d'expression de proteine WO2000039310A1 (fr)

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