EP1315822A2 - Vecteurs d'expression comprenant un fragment modifie de l'operon tryptophane - Google Patents

Vecteurs d'expression comprenant un fragment modifie de l'operon tryptophane

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Publication number
EP1315822A2
EP1315822A2 EP01947565A EP01947565A EP1315822A2 EP 1315822 A2 EP1315822 A2 EP 1315822A2 EP 01947565 A EP01947565 A EP 01947565A EP 01947565 A EP01947565 A EP 01947565A EP 1315822 A2 EP1315822 A2 EP 1315822A2
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EP
European Patent Office
Prior art keywords
recombinant protein
sequence
interest
mutated
sequence seq
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EP01947565A
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German (de)
English (en)
French (fr)
Inventor
Laurent Chevalet
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Pierre Fabre Medicament SA
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Pierre Fabre Medicament SA
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Application filed by Pierre Fabre Medicament SA filed Critical Pierre Fabre Medicament SA
Publication of EP1315822A2 publication Critical patent/EP1315822A2/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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C12N15/71—Expression systems using regulatory sequences derived from the trp-operon

Definitions

  • the invention relates to a construct for the expression of a gene coding for a recombinant protein of interest placed under the control of the tryptophan Ptrp operon in a prokaryotic host cell, which comprises directly downstream of the initiation codon a sequence nucleic acid with sequence SEQ ID No. 1 and downstream of this sequence a multiple cloning cassette intended to receive the gene coding for said recombinant protein of interest, at least one of the nucleotides of the sequence SEQ ID No. 1 being mutated or deleted so as to allow an overexpression of said recombinant protein.
  • the subject of the invention is also a vector containing such a construct, a prokaryotic host cell transformed by said vector, as is a method for producing a recombinant protein of interest using a construct according to the invention.
  • Bacterial cells are privileged hosts for the expression of recombinant proteins because they have limited nutritional requirements while being able to reach high growth densities, but also because they have been the subject of in the past. numerous investigations which have led to the generation of mutants of interest and various plasmid expression systems.
  • Escherichia coli E. coli
  • E. coli is the most used and best characterized organism if we judge by the abundant literature relating to the expression of proteins of prokaryotic or eukaryotic origin.
  • a messenger RNA must contain a sequence specifying the binding of the bacterial ribosome and allowing the initiation of translation. This sequence, called the Ribosome Binding Site (RBS), is located in an area covering the initiator codon.
  • RBS Ribosome Binding Site
  • Adenine-rich motifs immediately downstream of the initiation codon are favorable for the initiation of translation (Scherer, GFE, et al., Nucleic Acids Research, 8, 3895-3907, 1980; Chen, H., et al ., Journal of Molecular Biology, 240, 20-27, 1994b).
  • the AAA and GCU codons which are most frequent in the position of second codons Gold, L., 1988
  • GUIG or UUG sub-optimal
  • the present invention demonstrates the advantage in terms of translational efficiency of new nucleotide sequences carried by an expression vector, in the region of the "Ribosome Binding Site" (RBS), downstream of the tryptophan promoter (Ptrp).
  • RBS Ribosome Binding Site
  • Ptrp tryptophan promoter
  • CAT chloramphemcol acetyl transferase
  • a subject of the present invention is thus a construction for the expression of a gene coding for a recombinant protein of interest placed under the control of the promoter of the tryptophan Ptrp operon in a prokaryotic host cell comprising directly downstream of the codon of initiation of a nucleic sequence of sequence SEQ ID No. 1, and downstream of this sequence a multiple cloning cassette intended to receive the gene coding for said recombinant protein of interest, characterized in that at least one of the nucleotides of the sequence SEQ ID No. 1 is mutated or deleted so as to allow an overexpression of said recombinant protein.
  • recombinant protein of interest is intended to denote any protein, polypeptide or peptide obtained by genetic recombination, and capable of being used in fields such as that of human or animal health, cosmetology, animal nutrition, agro-industry or the chemical industry.
  • proteins of interest there may be mentioned in particular, but not limited to: - a cytokine and in particular an interleukin, an interferon, a tissue necrosis factor and a growth factor and in particular hematopoietic (G-CSF, GM - CSF), a human growth hormone or insulin, a neuropeptide;
  • a factor or cofactor involved in coagulation and in particular the NUI factor, von Willebrand factor, antithrombin III, protein C, thrombin and hirudin; - an enzyme and in particular trypsin, a ribonuclease and ⁇ -galactosidase;
  • an enzyme inhibitor such as ⁇ 1 antitrypsin and viral protease inhibitors
  • - a protein capable of inhibiting the initiation or progression of cancers, such as the expression products of tumor suppressor genes, for example the P53 gene; - a protein capable of stimulating an immune response or an antigen, such as for example the proteins, or their active fragments, of the membrane of bacteria Gram negative, in particular OmpA proteins from Klebsellia or protein G from human respiratory syncytial virus;
  • a protein capable of inhibiting a viral infection or its development for example the antigenic epitopes of the virus in question or altered variants of viral proteins capable of entering into competition with the native viral proteins;
  • - a protein capable of being contained in a cosmetic composition such as substance P or a superoxide dismutase; - a food protein and in particular a food;
  • nucleic sequence of sequence SEQ ID ⁇ ° 1 in which at least one of the nucleic acids is mutated or deleted so as to allow an overexpression of said recombinant protein is meant any sequence which comprises a deletion or a mutation of at least one nucleotide of the sequence SEQ ID N ° 1 which allows an overexpression of the recombinant protein, compared to the expression of said recombinant protein obtained using the sequence SEQ ID N ° 1 unmodified.
  • deletion is meant the elimination of one or more nucleotides at one or more nucleotide sites of the sequence SEQ ID No. 1.
  • the resulting sequence is shortened compared to that of origin.
  • mutation is meant the replacement of one nucleotide by another (A by C, G or T; C by A, G or T; G by A, C or T; T by A, C or G).
  • the resulting sequence is the same size as the original.
  • the overexpression that is to say the fact of obtaining an expression greater than that obtained without the modification downstream of the initiation codon can be determined in particular by using one of the following methods: i) migration by SDS-PAGE of the total proteins of the bacterium and revelation of the recombinant protein by staining with Comassie Blue or by Western blot; ii) assaying the recombinant protein by a method involving a specific antibody (Elisa); iii) enzymatic assay if the recombinant protein has a catalytic activity.
  • method ii) is used, detailed in Example III.
  • multiple cloning cassette is meant a nucleotide sequence containing one or more restriction sites, which sites can be used during steps of cloning the gene of interest downstream of the start codon.
  • said at least nucleotide of the sequence SEQ ID No. 1 is deleted so as to allow overexpression of said recombinant protein.
  • the invention also relates to a construction according to the invention in which said at least mutated or deleted nucleotide, preferably deleted, is located on the fragment of sequence SEQ ID No. 2 of the sequence SEQ ID No. 1. Another object of the invention The invention relates to the constructions in which said at least mutated or deleted nucleotide, preferably mutated, is located on the codon GTA and / or on the codon GCA and / or on the codon CTG of the sequence SEQ ID No. 1.
  • said sequence SEQ ID No. 1 of which at least one of the nucleotides is mutated or deleted has at least in position 1, 2 and 3, the nucleotide A.
  • At least one of the nucleotides, and preferably all the nucleotides, located between the nucleic sequence of sequence SEQ ID No 1 and the multiple cloning cassette intended to receive the gene coding for said protein recombinant of interest are deleted.
  • said sequence SEQ ID No. 1 of which at least one of the nucleic acids is mutated or deleted and all the nucleotides located between the nucleic sequence of sequence SEQ ID No. 1 and the multiple cloning cassette are completely deleted, so that the initiation codon is directly upstream of the multiple cloning cassette.
  • the constructs contain a nucleic sequence directly upstream of the chosen initiation codon among the sequences of sequence SEQ ID N ° 3, SEQ ID N ° 4, SEQ ID N ° 5, SEQ ID N ° 6, SEQ ID N ° 7, SEQ ID N ° 8, SEQ ID N ° 9 and SEQ ID N ° 10.
  • the invention comprises a construction according to the invention, characterized in that the prokaryotic host cell is a gram negative bacterium, preferably belonging to the species E. coli.
  • Another object of the invention relates to a vector containing a construct as defined above, just like a prokaryotic host cell, preferably belonging to the species E. coli, transformed by such a vector.
  • the present invention also relates to a process for the production of a recombinant protein of interest in a host cell using a construct as defined above.
  • the present invention also relates to a process for the production of a recombinant protein of interest according to the invention, in which said construct is introduced into a prokaryotic host cell, preferably by a vector as defined above.
  • a process for the production of a recombinant protein of interest according to the invention is preferred, characterized in that it comprises the following steps: a) cloning of a gene of interest in a vector according to the invention; b) transformation of a prokaryotic cell with a vector containing a gene coding for said recombinant protein of interest; c) culturing said transformed cell in a culture medium allowing expression of the recombinant protein; and d) recovering the recombinant protein from the culture medium or said transformed cell.
  • the invention further comprises a use of a prokaryotic construct, vector or host cell according to the present invention, for the production of a recombinant protein.
  • the invention relates to a use of a recombinant protein for the preparation of a medicament intended for administration to a patient in need of such treatment, characterized in that said recombinant protein is produced by a process for the production of a protein recombinant of interest according to the invention.
  • Figure 1 Map of the plasmid vector pTEXmpl ⁇ and sequence SEQ ID N ° 39 of the region 1-450 comprising the promoter / operator Ptrp, the region of the leader TrpL, the multiple cloning site mpl8 and the terminator of transcription.
  • Figure 2 Restriction map of the RBS region (Ribosome Binding Site) on the vector pTEXmpl 8 (SEQ ID No. 40).
  • Figure 3 Estimation on SDS-PAGE gel of CAT expression in bacteria transformed by the pTEXCAT or pTEXCAT4 vectors.
  • Figure 4 Comparative study of the expression of ⁇ -galactosidase from the vectors pTEX- ⁇ GAL and pTEX4- ⁇ GAL (kinetics in fermenter).
  • This example illustrates one of the aspects which led to the invention, and in particular the way in which the library of plasmid vectors carrying the tryptophan Ptrp promoter and randomly mutated upstream of the initiation codon is constructed.
  • the original vector is described in FIG. 1. It is a plasmid derived from pBR322 (Bolivar, F., et al., Gene, 2, 95-113, 1977) in which the promoter / operator Ptrp (1-298), followed by the sequence coding for the first 7 amino acids of the leader TrpL from E.
  • the reporter gene for chloramphenicol acetyl transferase is cloned at the ⁇ coRI and PstI sites of pTEXmpl 8.
  • the coding sequence of the cat gene is amplified by PCR at 1 using CATfor and CATrev oligonucleotides whose sequences are: CATfor: 5 '-CCGGAATTCATGGAGAAAAAAATCACTGG-3' (SEQ ID N ° 11)
  • SK (Stratagene, La Jolla, CA, USA).
  • the amplification product is deposited on agarose gel and purified according to the GeneClean method (BiolOl, La Jolla, CA).
  • the cloning of the insert in pTEXmpl 8 is verified after transformation in E. coli by the appearance of colonies developing on boxes of LB agar medium (Sambrook J., et al., Molecular cloning. A laboratory manual, 2 nd edition Plainview, NY: Cold Spring Harbor Laboratory Press, 1989) in the presence of 30 ⁇ g / ml of chloramphenicol.
  • the sequence of the insert is confirmed by automatic sequencing using the “Dye Terminator” kit and the DNA sequencer 373A (Perkin ⁇ lmer Applied Biosystems, Foster City, CA).
  • the vector obtained is named pT ⁇ XCAT.
  • the insertion of RBS having a degenerate sequence upstream of the initiation codon is carried out by ligation of synthetic oligonucleotides at the Spel and ⁇ coRI sites of the vector pT ⁇ XCAT.
  • the region going from the Spel site to the ⁇ coRI site respectively in positions - 49 and + 28 (see FIG. 2) is deleted by enzymatic digestion and replaced by a heteroduplex formed by two partially degenerate synthetic oligonucleotides, hybrids between them.
  • oligonucleotides RanSDl / RanSD2 and RanSD3 / RanSD4 Two pairs of oligonucleotides are used, involving respectively the oligonucleotides RanSDl / RanSD2 and RanSD3 / RanSD4 whose sequences are: RanSDl: 5'CTAGTTAACTAGTACGCAAGTTCACGTAAANNNNNNNNNNNNNÎWNNNATG AAAGCAATTTTCGTACTGAATGCGG-3 '(S ⁇ Q ID NO: 13) RanSD2:
  • the four oligonucleotides were synthesized by MWG Biotech (Ebersberg, D) under conditions ensuring an equimolar distribution of the bases for each degeneration.
  • the number of combinations (4 16 or approximately 4.3 10 9 ) allows the screening of RBS which are optimized both from the point of view of their Shine-Dalgarno (SD) sequence, of the sequence located between the SD region and the d codon. initiation as well as in SD-ATG spacing. This library will be named (N 16 ) in the remainder of the text.
  • the RanSD3 / RanSD4 pair provides complete degeneration on 6 nucleotides preceding PATG and partial degeneration on the 7 nucleotides upstream.
  • This second library is named (RN 6 ).
  • the linearization of the vector pTEXCAT and its purification on gel, the hybridization of the oligonucleotides in pairs, the ligation of the heteroduplexes to the linearized pTEXCAT vector and the transformation in E. coli of the library thus constituted are carried out according to the conditions described by Sambrook J., et al. (1989). Conventionally, 100 fmol of vector and 1000 fmol of insert are used in a ligation reaction in the presence of T4 ligase in a final volume of 15 ⁇ l. The reaction is carried out overnight at 16 ° C.
  • Electrocompetent TOP 10 bacteria (50 ⁇ l) are then transformed by electroporation with 3 ⁇ l of the ligation mixture under the conditions recommended by the supplier (Invitrogen, CarIsbad, CA).
  • the transformation mixture is spread on LB agar dishes containing 200 ⁇ g / ml of ampicillin, giving rise after 16 hours of incubation at 37 ° C. to the appearance of transformed colonies.
  • Example II
  • the figures (upper row) indicate the number of colonies counted after 18 h of incubation at 37 ° C., each medium having been seeded with approximately 100 cells.
  • CA transformed by the vector pTEXCAT and spread on dishes containing different concentrations of chloramphenicol have, between 300 and 600 ⁇ g / ml of chloramphenicol, a stronger development in the presence of 3- ⁇ indole acrylic acid (LAA), an analogue of tryptophan acting as an inducer by a Ptrp derepressure effect (Marmorstein, RQ and Sigler, PB, The Journal of Biological Chemistry, 264, 9149-9154, 1989).
  • LAA 3- ⁇ indole acrylic acid
  • Ptrp derepressure effect Marmorstein, RQ and Sigler, PB, The Journal of Biological Chemistry, 264, 9149-9154, 1989.
  • This example illustrates the selection of clones from the libraries constructed according to the description of Example 1.
  • the libraries obtained in the form of colony mats on boxes of LB agar + ampicillin are taken up in sterile water so as to reconstitute a suspension whose Optical Density (OD) at 580 nm is close to 1.
  • this suspension is spread on LB agar dishes containing lethal doses of chloramphenicol (600, 700, 800 and 900 ⁇ g / ml) at a rate of 100 ⁇ l of suspension per Petri dish. The dishes are incubated at 37 ° C.
  • the clones selected at this stage are then subjected to a series of analyzes: (i) extraction of the plasmid (Qiagen kit, Hilden, D) and sequencing of the region covering the RBS, (ii) culture in Erlenmeyers with induction by 1TAA then estimation of the level of expression of CAT by ELISA assay, (iii) SDS-PAGE electrophoresis of the total proteins extracted from the previous cultures and staining with Coomassie blue making it possible to visualize the total intracellular proteins.
  • the sequencing of the clones is carried out using the Dye Terminator kit on an ABI 373A sequencer (Perkin Elmer Applied Biosystems, Foster City, CA).
  • Erlenmeyer cultures are produced by inoculating 25 ml of TSBY medium (Tryptic Soy Broth (DIFCO) 30 g / 1 + Yeast Extract (Difco) 5 g / 1) + tetracycline 8 mg / 1 by a colony on a dish or by a suspension bacterial stored at - 80 ° C. Each preculture is incubated on a plate stirred at 200 rpm and 37 ° C overnight. A fraction is transferred into 50 ml of the same medium so as to reach an initial optical density equal to 1. For the induction of the CAT protein, the medium is added with 25 mg / 1 of IAA and then stirred in the same conditions for 5 hours.
  • TSBY medium Traptic Soy Broth (DIFCO) 30 g / 1 + Yeast Extract (Difco) 5 g / 1) + tetracycline 8 mg / 1 by a colony on a dish or by a suspension bacterial stored at - 80
  • a fraction of the suspension (3 x 1 ml diluted to OD 0.1) is centrifuged and the cells stored at ⁇ 20 ° C. for the CAT assay by ELISA (CAT ELISA kit, Roche Diagnostics, Basel, CH). The rest of the biomass is recovered by centrifugation at 10,000 g, 4 ° C for 15 minutes. The biomass is taken up in a TEL buffer (25 mM Tris, 1 mM EDTA, Lysozyme 500 ⁇ g / ml, pH 8) at a rate of 5 ml per 1 g of wet biomass.
  • TEL buffer 25 mM Tris, 1 mM EDTA, Lysozyme 500 ⁇ g / ml, pH 8
  • the cells are lysed by sonication (NibraCell sonicator equipped with a micro-probe, Sonics & Materials, Danbury, CT).
  • sonication NebraCell sonicator equipped with a micro-probe, Sonics & Materials, Danbury, CT.
  • One ml of the resulting suspension is centrifuged for 5 min at 12,000 rpm.
  • the pellet is taken up in 200 ⁇ l of TEL to give the insoluble fraction (I).
  • the supernatant is noted “S”.
  • the total proteins contained in fractions I and S are analyzed by electrophoresis under denaturing conditions (SDS-PAGE) and staining with Coomassie blue.
  • Table 2 below indicates the different RBS sequences obtained after screening the two libraries (N 16 ) and (R 7 N 6 ). Following alignment in the GenBank and EMBL nucleotide databases, we can conclude that none of the sequences of 16 nucleotides (strategy (N 16 )) or 13 nucleotides (strategy (R 7 N 6 )) located immediately upstream of the AUG codon in the various isolated clones has not been described to date.
  • Each nucleotide sequence (messenger RNA) comprises the mutated region downstream of the initiation codon.
  • the reference sequence of the pTEXCAT vector is shown in the first line of the table.
  • Nucleic sequences upstream and downstream of the codon initiation vectors are shown in this table after transcription as RNA. At the 3 'end of these sequences, only the first two codons of the multiple cloning site are represented, namely GAAUUC.
  • the clones described in Table 2 have mutations in the region of RBS situated immediately downstream of the codon AUG.
  • the clones pTEXCAT4, pTEXCATl 'and pTEXCAT3' carry a point mutation affecting an amino acid of the N-terminal part of the encoded protein (respectively Leu7Pro, Ala3Pro and Val ⁇ Ala).
  • the other clones carry larger rearrangements: pTEXCAT2 ', pTEXCAT5' and pTEXCAT9 'have deletions inducing the loss of the regions Ala3Leu7, Ile4Leu7 and Ile4Asn8 respectively.
  • the vector pTEXCAT4 was reconstructed in vitro from pTEXCAT by Spel-EcoRI digestion and ligation of a duplex formed by the two.
  • the resulting vector noted pTEXCAT-SD4 was then transformed into E. coli TOP 10 and compared to pT ⁇ XCAT4 in terms of potential for expression of the CAT enzyme.
  • the results obtained indicate that the expression levels of pT ⁇ XCAT4 and pT ⁇ XCAT-SD4 are comparable to each other and significantly higher than pT ⁇ XCAT. This supports the hypothesis that the improvement in expression observed with the clones claimed in this patent application is indeed caused specifically by the sequences located between the Spel and ⁇ coRI sites.
  • the two vectors were transformed into the E. coli ICONE 200 strain (French patent application FR 2 777 292 published on October 15, 1999) with a view to culturing in a fermenter with kinetic monitoring of the expression of ⁇ -galactosidase .
  • the recombinant bacteria ICONE 200 x pTEX- ⁇ GAL and ICONE 200 x pTEX4- ⁇ GAL were cultured in 200 ml of complete medium (Tryptic Soy Broth (DIFCO) 30 g / 1, Yeast Extract (DIFCO) 5 g / 1) for overnight at 37 ° C.
  • the cell suspension obtained was transferred sterile to a fermenter (CF3000 model from Chemap, capacity 3.5 1) containing 1.8 liters of the following medium (concentrations for 2 liters of final culture): glycerol 90 g / 1, (NH_ ⁇ ) 2S04 5 g / 1, KH2PO4 6 g / 1, K2HPO4 4 g / 1, Na3-citrate 2H2O 9 g / 1, MgS 4 7H2O 2 g / 1, yeast extract 1 g / 1, trace elements, defoamer 0.06% , tetracycline 8 mg / 1, tryptophan 200 mg / 1.
  • the pH is adjusted to 7.0 by adding ammonia.
  • the dissolved oxygen rate is maintained at 30% of saturation by slaving the stirring speed and then the aeration rate to measure dissolved PO2.
  • the induction is carried out by adding 25 mg / 1 of IAA (Sigma, St Louis, MO).
  • Kinetic analysis of the optical density of the culture (OD to 580 nm) and intracellular ⁇ -galactosidase activity was performed.
  • the level of ⁇ -galactosidase activity is estimated by a colorimetric assay by mixing 30 ⁇ l of sample (fraction “S”, see example 3), 204 ⁇ l of buffer (Tris-HCl 50 mM pH 7.5 - MgCl 2 1 mM) and 66 ⁇ l of ONPG (4 mg / ml in 50 mM Tris-HCl pH 7.5). The reaction mixture is incubated at 37 ° C. The reaction is stopped by the addition of 500 ⁇ l of Na 2 CO 3 1 M. The OD at 420 nm relative to the incubation time is proportional to the ⁇ -galactosidase activity present in the sample. Knowing that E.
  • coli ICONE 200 has a complete deletion of Lac Popon, the ⁇ -galactosidase activity measured is only due to the expression of the lacZ plasmid gene.
  • the results of this comparative study indicate that in two independent experiments, the vector pTEX4- ⁇ GAL gives a level of ⁇ -galactosidase activity approximately 50 times higher than pTEX- ⁇ GAL ( Figure 4).
  • Figure 4 We deduce that the original sequence isolated in the RBS area of the vector pTEXCAT4 potentiates the expression, not only of the CAT protein, but also of other proteins such as, for example, ⁇ -galactosidase.
  • the vectors pTEXIO ', pTEXl P and pTEX12' originate from the vector pTEX9 but also include additional mutations, as indicated in table 4 below: Table 4. Comparison between the expression levels given by the vectors pTEXwt, pTEX9 *, pTEXIO ', pTEXl P and pTEX12'.
  • Each nucleotide sequence (messenger RNA) comprises the mutated region downstream of the initiation codon.
  • the reference sequence of the pTEXCAT vector is shown in the first line of the table.
  • the nucleic acid sequences upstream and downstream of the vector initiation codon are represented in this table after transcription in the form of RNA. At the 3 'end of these sequences, only the first two codons of the multiple cloning site are represented, namely GAAUUC.

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EP01947565A 2000-06-22 2001-06-21 Vecteurs d'expression comprenant un fragment modifie de l'operon tryptophane Withdrawn EP1315822A2 (fr)

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FR0008002A FR2810675B1 (fr) 2000-06-22 2000-06-22 Construction modifiee en aval du codon d'initiation pour la surexpression de proteines recombinantes
PCT/FR2001/001952 WO2001098453A2 (fr) 2000-06-22 2001-06-21 Vecteurs d'expression comprenant un fragment modifie de l'operon tryptophane

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EP2185704A1 (en) * 2007-08-03 2010-05-19 Pfenex, Inc. Translation initiation region sequences for the optimal expression of heterologous proteins
KR100961565B1 (ko) * 2008-03-10 2010-06-07 충남대학교산학협력단 목적하는 단백질 발현 수준을 제공하는 초기 코돈의발굴방법, 및 이를 이용한 재조합 단백질의 발현 조절 방법및 생산 방법
US20110129873A1 (en) * 2008-04-30 2011-06-02 Monsanto Technology Llc Recombinant DNA Vectors for Expression of Human Prolactin Antagonists
US20110111977A1 (en) * 2008-07-03 2011-05-12 Pfenex, Inc. High throughput screening method and use thereof to identify a production platform for a multifunctional binding protein
CN110491447B (zh) * 2019-08-05 2021-08-17 浙江省农业科学院 一种用于异源基因体外表达的密码子优化方法及应用
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US20040260060A1 (en) 2004-12-23
BR0111907A (pt) 2003-12-30
WO2001098453A3 (fr) 2002-08-01
AU6922401A (en) 2002-01-02
FR2810675A1 (fr) 2001-12-28
CN1443242A (zh) 2003-09-17
WO2001098453A2 (fr) 2001-12-27
MXPA02012880A (es) 2003-05-14

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