WO2001029225A1 - Procede general permettant d'ameliorer l'expression des proteines heterologues - Google Patents

Procede general permettant d'ameliorer l'expression des proteines heterologues Download PDF

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WO2001029225A1
WO2001029225A1 PCT/US2000/008477 US0008477W WO0129225A1 WO 2001029225 A1 WO2001029225 A1 WO 2001029225A1 US 0008477 W US0008477 W US 0008477W WO 0129225 A1 WO0129225 A1 WO 0129225A1
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protein
host cells
expression
selector
fusion
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PCT/US2000/008477
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Robert F. Balint
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Panorama Research, Inc.
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Priority to EP00921531A priority Critical patent/EP1226241A1/fr
Priority to CA002387646A priority patent/CA2387646A1/fr
Priority to AU41832/00A priority patent/AU4183200A/en
Publication of WO2001029225A1 publication Critical patent/WO2001029225A1/fr

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    • 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
    • C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Natural proteins have three fundamental properties in vivo which can be exploited to obtain stable expression of heterologous proteins in the absence of any distinguishing phenotype of the protein itself.
  • a fold is simply a minimum energy conformation or ground state.
  • the vast majority of sequences in protein sequence space do not have unique folds, but rather have multiple, inter-convertible minimum energy conformations (Li et al., Science ( 1996) 273:666-669; Godzik, TIBTECH (1997) 15: 147-151 ; Sauer, Folding and Design ( 1996) 1 :R27-R30; Govindarajan and Goldstein, Proc. Natl. Acad. Sci USA (1996) 93:3341-3345).
  • a multi-domain protein is only as stable in vivo as its least stable domain.
  • nascent proteins first encounter the hsp40 and hsp70 classes of chaperone proteins and their associates which bind to any exposed hydrophobic sequence to protect the nascent protein in its unfolded state (Hartl, Nature (1996) 381 :571-580). The new protein is then released from the chaperones by a cooperative, energy-dependant mechanism. Many proteins then fold with two-state kinetics, collapsing rapidly into their native folds without discernable intermediates. The remainder, however, may accumulate as 'molten globule' intermediates while searching for conformation space for their native folds.
  • any protein, new or old, which undergoes sufficient transient thermal or chemical denaturation to expose hydrophobic surface may be bound and unfolded by the chaperonin complex.
  • Each protein may then undergo multiple rounds of binding, unfolding, release, and refolding until its native fold is achieved. However, after each round in which a protein still fails to achieve its native fold, it may either be rebound by the folding complex, or it may be bound by the protein turnover machinery, which also recognizes exposed hydrophobic surfaces.
  • These alternative fates for nascent proteins are illustrated in Figure 1. Thus, the longer it takes a protein to fold, the more vulnerable it is to proteolysis or aggregation.
  • Natural proteins cannot be expected to fold any more efficiently than necessary in their natural milieus.
  • eukaryotic proteins may fold more slowly than prokaryotic proteins because the risk of aggregation is much greater in the prokaryotic cytoplasm. Because of the ten-fold higher prokaryotic protein synthesis rate, local concentrations of nascent proteins are much higher and nascent proteins have little chance to fold while still tethered to the ribosome.
  • solubility of the fusion protein may become limited by the folding rate of the marker domain, the solubility of the optimized protein of interest may be even higher when expressed alone, without the marker fusion domain.
  • rate-limiting intermediates may be readily destabilized by single mutations. This means that in mutagenic libraries with mutation frequencies on the order of one per molecule, the frequency of faster folders may be greater than ⁇ one-tenth of the inverse of the chain length, or ⁇ one in 2500 for a 250-residue protein.
  • large libraries are not needed to find high-expressing variants of poor expressors.
  • folding can be optimized with few mutations also minimizes the likelihood of introducing immunogenic epitopes into therapeutic proteins.
  • folding efficiency in vivo is not known, but with the subject invention, it may be possible to test those limits.
  • selectable markers can be optimized by mutagenesis and selection for maximum strength of phenotype. If such folding-optimized markers have any remaining tendency to aggregate when over-expressed, it will be even further reduced when they are expressed as fusions to mutagenized proteins of interest. Thus, folding-optimized markers should place no limit on the optimization of proteins of interest. This could allow valuable proteins to be produced in higher yields with higher activities and purity than previously possible.
  • the first relates to tolerance of extreme conditions, and the second relates to half-life under favorable conditions. They are not necessarily mutually inclusive. The reason for this is that activity is often lost reversibly before it is lost irreversibly, but the reverse is not possible. In fact, if loss of activity under extreme conditions were entirely reversible, it would have little to do with the half-life of the protein, which is primarily a function of the rate of irreversible aggregation. Each trait is potentially valuable for industrial proteins. Proteins which work better under extreme conditions, and/or last longer will fetch a premium on the market, in addition to savings realized from reduced production costs, and possible premiums for higher purity. Folding optimization selects primarily for reduced tendency to aggregate.
  • Methods are provided for obtaining host cells expressing a mutant of a desired protein optimized for expression in the host cells, for obtaining a protein with enhanced stability as compared to a wild type of the desired protein, and for identifying peptides that can stabilize an unstable protein, in each case by expressing the protein linked to a selector protein that confers a selectable phenotype on the host cell.
  • the unstable protein is coexpressed with members of a random peptide library.
  • the method includes the steps of preparing a library of mutagenized coding sequences for the protein of interest, purifying the members of the library of mutagenized coding sequences, ligating each member of the library into an expression cassette in frame with the coding sequence for a selector protein, transforming a multiplicity of host cells with the expression cassettes, growing the resulting transformed host cells under conditions for which the selector protein confers ability for the transformed cells to grow to produce the mutant proteins joined to the selector protein, identifying cells that express mutant proteins at a selective pressure higher than that of cells expressing an unmutagenized protein.
  • Proteins with enhanced stability can be obtained by cleavage from the selector protein, or expressing the mutant protein as a free protein in the host cells for which it is optimized.
  • the invention finds use for example in optimizing mammalian peptides for improved expression in prokaryotic cells and for identifying peptides that can be used for treating diseases that are characterized by production of an unstable variant of a wild type protein.
  • Figure 1 An illustration of the alternative fates for nascent proteins when expressed in cells at normal levels. When overexpressed, aggregation is an additional fate (not shown).
  • DnaK and DnaJ are bacterial Hsp70 and Hsp40 proteins, respectively.
  • GroEL is the bacterial Hsp60 complex, and GroES is the companion HsplO complex.
  • GFP-CAT For 2-domain proteins like GFP-CAT, we hypothesize that misfolding of a single domain leads to turnover of the entire protein.
  • FIG. 1 Expression construct for GFP-CAT fusions.
  • T7prom phage T7 promoter;
  • G S flexible spacer between the GFP and CAT domains;
  • His ⁇ hexa-histidine tail for affinity purification;
  • T7t phage T7 transcription terminator;
  • ori origin of replication;
  • bla ampicillin resistance.
  • FIG. 1 Chloramphenicol resistance of E. coll NovaBlue DE3 cells expressing CAT, wtGFP-CAT, and GFPuv-CAT. Cells expressing each construct were plated at 1000 cells per plate onto solid LB medium containing 0.02 mM IPTG and icreasing concentrations of chloramphenicol. After overnight growth at 37° C colonies per plate were scored and plotted against cam concentration.
  • Figure 6. Selection of protein-stabilizing peptides from random peptide libraries (RPL) using the Fold Selector system.
  • Genes for unstable extra-cellular proteins of choice such as amyloid ⁇ protein (A ⁇ ), fused to the N-terminus of ⁇ -lactamase via a flexible linker, (G 4 S) 3 , may be transcribed from the trp-lac fusion promoter (trc prom) in a pl5A replicon (pl5A ori) with kanamycin resistance (kan) for plasmid retention.
  • trc prom trp-lac fusion promoter
  • kan kanamycin resistance
  • SP N-terminal signal peptides
  • the RPL genes encoding random peptides fused to the N-terminus of thioredoxin via a G 4 S linker, may be transcribed from the lac promoter in a pUC phagemid with chloramphenicol resistance (cat) for plasmid retention.
  • the phagemid origin of replication (fl ori) allows the RPL construct to be packaged in phage and quantitatively introduced into cells expressing the unstable protein by infection at high multiplicity (m.o.i.).
  • Peptides are selected by their ability to stabilize the p.o.c. and thereby confer growth on non- permissive antibiotic concentrations.
  • Methods for obtaining a protein of interest that is optimized for expression in a host cell are provided.
  • a protein that is optimized for expression in a particular host cell such as E. coli
  • members of a library of mutagenized coding sequences for the protein of interest joined to the coding sequence for a selector protein are transformed into the host cells which are then grown under conditions for which the selector protein confers ability for the transformed cells to grow.
  • the coding sequence for the chimeric protein contains a coding sequence for a linker peptide between the mutangenized library member and the coding sequence for the selector protein; prefereably the linker is a flexible linker.
  • pET23a is an ampicillin-resistant pBR322 derivative in which transcription of inserted coding sequences is controlled by the bacteriophage T7 promoter and transcription terminator (Moffatt and Studier, J. Mol. Biol. (1986) 189: 113-130). Expression is restricted to hosts, such as NovaBlue (DE3) (Novagen, Inc.), which have been transformed to express the T7 RNA polymerase.
  • GFP fluorescence can be readily observed in colonies of these cells harboring the pET-GFP construct by illuminating with long-wave uv light.
  • the spectrum, quantum yield, and extinction coefficient of GFPuv do not differ appreciably from wtGFP, consistent with a difference of only three of 238 amino acids (Crameri et al. , Nature Biotechnology (1996) 14:315-9).
  • GFPuv produces 30-45 times more steady state fluorescence than does wtGFP.
  • the selectable marker coding sequence must be inserted downstream from that of the protein of interest to insure that selection is not favored by premature termination of the protein of interest.
  • the CAT coding sequence was inserted into the Xhol site of p ⁇ T23a in the same reading frame as the upstream GFPs. Between the two a 15-residue flexible, hydrophilic linker, (Gly 4 Ser) 3 , was encoded with convenient restriction sites for facile replacement of both GFP and CAT sequences. The CAT sequence terminates in a His ⁇ tail for facile purification.
  • This construct, pET23a-GFP-CAT is shown in Figure 2.
  • Table I and Figure 3 show the results of comparisons of the chloramphenicol resistance and fluorescence characteristics of wtGFP and GFPuv expressed alone and as C-terminal fusions with CAT from pET23a in E. coli strain BL21(DE3).
  • GFPuv produces ⁇ 30 times more steady state fluoresence than wtGFP, as determined by fluorometry of suspensions of equal numbers of cells from overnight growth on solid medium.
  • Maximum transcription normally requires induction of T7 polymerase expression with IPTG, but a low level of transcription occurs even in the absence of IPTG.
  • GFPuv produces ⁇ 30-fold higher fluorescence intensity. From this we conclude that GFPuv is much less prone to aggregation at similar expression levels, i.e., nascent protein concentrations, presumably due to a higher folding rate.
  • Chloramphenicol resistance was determined as the highest concentration in solid LB medium on which at least 50% of cells plated formed visible colonies after overnight growth.
  • the GFP coding sequence may be subjected to random mutagenesis by any of several methods, including error-prone PCR (Cadwell and Joyce, in PCR Primer A Laboratory 20 Manual, Dieffenbach and Dveksler (eds.) (1995) Cold Spring Harbor Press, Cold Spring
  • the ligation product was then introduced into cells of E. coli strain NovaBlue (DE3) by high- voltage electroporation. Transformants were then plated onto solid Luria-Bertani medium containing increasing amounts of chloramphenicol, ranging from 34 ⁇ g/ml to 544 ⁇ g/ml, and incubated at 37°C overnight.
  • An initial assessment of the correlation of cam resistance with fluorescence intensity was made by a visual estimation of the percentage of colonies which fluoresced more intensely than wtGFP-CAT as a function of cam concentration. The results are illustrated in Figure 4. As the concentration of cam increased, the frequency of brighter colonies also increased.
  • OD ⁇ oo of 0.1 OD ⁇ oo of 0.1
  • Fluorescence emission spectra were then determined for the brightest mutants from each round, designated GFPR1-CAT and GFPR2-CAT respectively. Each had an emission maximum at 510 nm when excited at 390 nm. The emission spectra of these clones are compared to those of wtGFP-CAT and GFPuv-CAT in Figure 5.
  • the brightest mutant from round one, GFPR1-CAT was 14 times brighter than wtGFP-CAT and 3.5 times brighter than GFPuv-CAT.
  • GFPR1-CAT was also resistant to at least 408 ⁇ g/ml cam, whereas GFPuv-CAT could resist only 340 ⁇ g/ml.
  • the brightest mutant from round two, GFPR2-CAT was 56 times brighter than GFPwt and 14 times brighter than GFPuv-CAT, and could grow in 510 ⁇ g/ml cam.
  • the dramatic increases in expression seen with GFPR1-CAT and with GFPR2-CAT over GFPuv-CAT essentially vanished when CAT was removed.
  • GFPuv-CAT Rather the reduced expression of GFPuv-CAT is probably due to mutual steric interference with the folding of the two proteins, and the same is probably true to a lesser extent for GFPR1-CAT.
  • SDS-PAGE confirmed that the GFPR1-CAT, GFPR2-CAT, GFPwt-CAT, and GFPuv-CAT all comprised over 50% of the total cell protein.
  • the increase in brightness for the GFPR1-CAT and GFPR2-CAT mutants over that of wtGFP-CAT and GFPuv-CAT is reflected by the difference in the amount of protein in the soluble fraction. For example, about 25% of GFPR2-CAT protein is soluble, whereas only about 1-2% of wtGFP-CAT protein was soluble.
  • DNA sequences were determined for the entire open reading frames of GFPR1 and GFPR2, and compared to those of wtGFP and GFPuv (see Table III). In addition, the reported mutations in GFPuv were confirmed. Surprisingly, one mutation was shared by all three improved GFPs, V 164A. Even more surprisingly, this was the only mutation present in GFPR1. Since GFPR1 expresses as well or better than GFPuv as the free protein, this suggests the other mutations in GFPuv are not necessary.
  • GFPuv had originally been "evolved” by repeated rounds of recombinatorial mutagenesis by DNA shuffling and phenotypic selection, followed by back-crossing to eliminate deleterious mutations (Crameri et al.. Nature Biotechnology (1996) 14:315-9). However, it appears that only one of the three remaining mutations is actually required for the complete phenotype. Thus, not only was recombination unnecessary, but the required mutation could have been recovered easily from a few thousand clones of a standard Taq polymerase amplification of the wtGFP coding sequence.
  • V164A Since the V164A mutation could account for all of the increase in free GFP expression for all three improved GFPs, we wished to see if any other independently adaptive mutations could be recovered.
  • V164A appeared to be the only single-hit mutation capable of destabilizing the aggregation-prone intermediate in GFP folding. Indeed, such a mutation would be expected to reduce the hydrophobicity at that position, and it is hydrophobicity which would be expected to drive aggregation. Any other independently adaptive mutation of comparable frequency should have appeared at least once.
  • GFPR2 contains only one mutation in addition to V164A, namely N105S. Thus, this mutation is apparently responsible for the complete elimination of folding interference between GFP and CAT. It is not likely that the combination of mutations in GFPR2 arose by recombination because V164A is apparently indispensable. Rather, the combination probably arose by simple addition of the N 105S mutation to V164A. We have confirmed that the N 105S mutation by itself is not sufficient to confer a selectable increment in cam resistance on GFP-CAT.
  • GFP-N105S can only be selected in proteins which already fold independently, like GFPR1.
  • GFPR2 has continued to fold independently of the fusion partner, neither inhibiting nor being inhibited by it.
  • GFPR2-NPT C-terminal fusion with neomycin phosphotransferase
  • both fluorescence and kanamycin resistance were at least as high as those of the free GFPR2 and the free NPT, respectively, whereas both functions were inhibited in the GFPuv-NPT fusion.
  • GFPuv was reported to express at 30-fold higher levels than wtGFP in mammalian cells, it may exhibit the same sensitivity to fusion expression in these cells as it does in bacteria.
  • GFPR2 is the subject of US Patent Application 60/160,461.
  • Unstable proteins can be stabilized by peptides selected from random peptide libraries. Many diseases are caused by unstable proteins, which fail to accumulate in biologically active form in cells or tissues due to one or more mutations which cause a delay in folding or which destabilize the active conformation such that the protein is prone to insoluble aggregation and/or proteolysis. There are two main types of unstable proteinopathies: those which cause disease by forming toxic insoluble aggregates, and those which cause disease by loss of function. The former are represented by amyloidogenic polypeptides such as the amyloid ⁇ protein (A ⁇ ), which forms insoluble amyloid fibrils in the brain (Li et al. , J. Leukocyte Biol.
  • a ⁇ amyloid ⁇ protein
  • Amyloid deposits can induce chronic inflammation and tissue damage, which are major etiologic components of Alzheimer's disease.
  • chemo-therapeutic strategies to counter the progress of amyloidogenesis and resultant tissue degeneration in Alzheimer's and other amyloidogenic proteinopathies.
  • Drugs which could interfere directly with the aggregation of the A ⁇ protein would be highly desirable.
  • amyloidogenic proteins such as the A ⁇ peptide do not produce screenable or selectable phenotypes, there is no conventional method to select for stabilization of these proteins.
  • the selectable phenotype is destabilized and can be used to select for stabilization of the amyloidogenic protein.
  • Extracellular amyloidogenic proteins may be expressed in the E. coli periplasm as C-terminal fusions to TEM-1 ⁇ -lactamase with an intervening flexible linker such as (Gly Ser) 3 .
  • TEM-1 ⁇ -lactamase is an E.
  • RPL random peptide-encoding library
  • At least 10 8 clones of the RPL were rescued as filamentous bacteriophage by infection with helper phage M13K07 (Sambrook et al , in Molecular Cloning A Laboratory Manual, 2 nd ed. , (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 4.19-4.50). At least 10 9 DH5 ⁇ cells bearing
  • the A ⁇ - ⁇ -lactamase construct were infected with a 100-fold excess of RPL phage to insure quantitative infection. At least 10 s independent transfectants were then plated onto solid medium containing 400, 600, and 800 ⁇ g/ml ampicillin. 110 colonies were recovered after overnight growth on 400 ⁇ g/ml, 19 were recovered from 600 ⁇ g/ml, and 4 were recovered from 800 ⁇ g/ml. For negative controls, ten clones were selected at random from the
  • stabilizing peptides may need to interact with residues which become iternalized in the active conformation. Also, the diversity of constructive interactions which can occur between unstable proteins and flexible peptides should be much greater than interactions with the surfaces of rigid proteins. Thus, some of the stabilizing peptides may extend into the interior of the stabilized A ⁇ protein, and/or may interact with non-contiguous regions of the protein. It is even possible that in cases where instability is due to folding intermediates, and not to a loss of stability of the active conformation, that stabilizing peptides may, in effect, catalyze the folding reaction without remaining structural components of the folded protein.
  • a ⁇ -stabilizing peptides will not require all 12 amino acids for activity, and may be equally active as smaller peptides.
  • the methods described herein can be broadly used to isolate peptides which can stabilize desired proteins, particularly those which do not produce screenable or selectable phenotypes.
  • the protein of choice is a secreted protein, as in the case of the A ⁇ peptide, ⁇ -lactamase may be used as the C-terminal fusion partner to allow periplasmic selection for ⁇ -lactam antibiotic resistance.
  • a signal peptide must be encoded at the N-terminus for export of the fusion protein to the bacterial periplasm. It is preferable to use the pl5A replicon with chloramphenicol resistance, so that universal RPLs can be constructed in the pUC phagemid with kanamycin resistance.
  • the protein of choice is cytoplasmic, as in the case of GFP described above, CAT (Genbank accession no.
  • X06403 Rose, Nucleic Acids Research (1988) 16:355
  • may be used as the fusion partner to allow selection for chloramphenicol resistance (Dekeyzer et al. , Protein Engineering (1994) 7: 125-130; Zelazny and Bibi, Biochemistry (1996) 35: 10872-10878).
  • the first requirement which must be met is that the unstable protein must cause a substantial quantitative reduction in the selectable phenotype. This must be quantified and the minimum stringency must be established for quantitative selection, as was done for the use of ampicillin resistance to select for stabilization of the A ⁇ protein.
  • One or more universal RPLs may then be quantitatively introduced into cells expressing the fusion of the desired protein with the selector, and the transfectants are then plated onto the minimum concentration of antibiotic which is quantitatively non-permissive for growth of the fusion protein.
  • the number of independent transformants plated should be equivalent to or greater than the size of the RPL, and the minimum non-permissive concentration of antibiotic should allow no colonies to grow from the same number of cells expressing the fusion protein alone.
  • the RPL used was a 12-mer on the N-terminus of thioredoxin, but the RPL may vary in length from 3 to 20 or more residues on either end of the carrier. However, the proportion of unstable peptides in the RPL rapidly increases when the length exceeds — 12 amino acids.
  • the carrier may be any stable protein which tolerates terminal fusions well. Selected peptides may be verified by co- expression with the free protein of choice. A substantial increase in the proportion of the protein which partitions into the soluble fraction should be observed in the presence of the selected peptide only and not in the presence of a non-selected peptide.
  • coli T ⁇ M- 1 ⁇ -lactamase (Sutcliffe, 1978, Proc Natl Acad Sc. USA 75, 3737-41) may be separated into two fragments at E 197-L198 which can complement to form active enzyme with the aid of interacting domains such as hetero-dimerizing helixes which are fused to the break-point termini of the fragments (Balint and Her, US Patent Application 60/124,339).
  • the activity of the ⁇ -lactamase fragment complementation system is limited, however, by the stability of the N-terminal fragment, denoted ⁇ l97.
  • ⁇ l97 and the stable C-terminal fragment, ⁇ l98 were co-expressed in the E. coli periplasm as fusions to the hetero-dimerizing helixes of the c-fos and c-jun subunits of the transcription factor AP- 1 (Karin et al.. 1997, Curr Opin Cell Biol 9. 240-6), only enough ⁇ -lactamase activity was produced to confer a plating efficiency of ⁇ ⁇ % on 50 ⁇ g/ml ampicillin.
  • the GRE tripeptide conferred no resistance to ampicillin in the absence of the interacting helixes, thus it does not stabilize the re-folded fragment complex, but rather it must stabilize the ⁇ l97 fragment since activity is limited by the amount of soluble ⁇ l97. Since the GRE tri-peptide had the same stabilizing effect on ⁇ l97 fragment when a different carrier was used, its activity must be context independent. Thus, an 18 kDa enzyme fragment could be stabilized at least 100-fold by a tri-peptide selected from a random sequence library.

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Abstract

L'invention concerne des procédés comprenant des variantes de protéines ne n'offrant aucun phénotype pouvant être sélectionné, mais pouvant néanmoins être sélectionnées pour une expression stable dans des hôtes hétérologues. L'invention concerne également des procédés associés comprenant des bibliothèques d'expressions de l'ADN complémentaire, pouvant être enrichies pour une expression stable des domaines de repliement autonome des chaînes dans des hôtes hétérologues. L'invention concerne également des procédés associés comprenant des peptides stabilisant des protéines instables, lesquels peptides peuvent être sélectionnés de manière aléatoire parmi des bibliothèques de peptides. Si une protéine hétérologue s'exprime par une fusion avec un phénotype pouvant être sélectionné, la résistance du phénotype est proportionnelle au taux de repliement des chaînes, et de ce fait à la solubilité de la protéine présentant un intérêt. Ainsi, le phénotype pouvant être sélectionné peut être utilisé pour sélectionner de meilleurs agents d'expression à partir des bibliothèques de protéines présentant un intérêt issues de la mutagenèse; ou encore, ce phénotype peut être utilisé pour sélectionner des domaines de repliement autonome de chaînes à partir de bibliothèques d'expressions de l'ADN complémentaire, ou pour sélectionner des peptides qui stabilisent des protéines instables.
PCT/US2000/008477 1999-10-21 2000-03-29 Procede general permettant d'ameliorer l'expression des proteines heterologues WO2001029225A1 (fr)

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EP00921531A EP1226241A1 (fr) 1999-10-21 2000-03-29 Procede general permettant d'ameliorer l'expression des proteines heterologues
CA002387646A CA2387646A1 (fr) 1999-10-21 2000-03-29 Procede general permettant d'ameliorer l'expression des proteines heterologues
AU41832/00A AU4183200A (en) 1999-10-21 2000-03-29 A general method for optimizing the expression of heterologous proteins

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US16046199P 1999-10-21 1999-10-21
US60/160,461 1999-10-21
US51009700A 2000-02-22 2000-02-22
US09/510,097 2000-02-22

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US7943375B2 (en) 1998-12-31 2011-05-17 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US8133494B2 (en) 2001-07-05 2012-03-13 Novartis Vaccine & Diagnostics Inc Expression cassettes endcoding HIV-1 south african subtype C modified ENV proteins with deletions in V1 and V2
US9598469B2 (en) 2001-07-05 2017-03-21 Novartis Vaccines And Diagnostics, Inc. HIV-1 south african subtype C env proteins
WO2004016648A1 (fr) * 2002-07-19 2004-02-26 Amaxa Gmbh Proteine fluorescente
WO2005111060A2 (fr) * 2004-05-17 2005-11-24 Gardner, Rebecca Molecules impliquees dans le repliement des proteines
WO2005111060A3 (fr) * 2004-05-17 2007-02-22 Gardner Rebecca Molecules impliquees dans le repliement des proteines
FR2886943A1 (fr) * 2005-06-10 2006-12-15 Biomethodes Sa Methode de selection de proteines stables dans des conditions physico-chimiques non standard
WO2006134240A1 (fr) * 2005-06-10 2006-12-21 Biomethodes Methode de selection dans des conditions physico-chimiques non standard de proteines stables
US9903873B2 (en) 2005-06-10 2018-02-27 Biomethodes Method for selecting stable proteins in non-standard physicochemical conditions

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