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WO2004050878A1 - Procede pour modifier l'expression de polypeptides dans des systemes d'expression recombinants - Google Patents

Procede pour modifier l'expression de polypeptides dans des systemes d'expression recombinants Download PDF

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Publication number
WO2004050878A1
WO2004050878A1 PCT/EP2003/013410 EP0313410W WO2004050878A1 WO 2004050878 A1 WO2004050878 A1 WO 2004050878A1 EP 0313410 W EP0313410 W EP 0313410W WO 2004050878 A1 WO2004050878 A1 WO 2004050878A1
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Prior art keywords
expression system
gene
expressed
codons
expression
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German (de)
English (en)
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Walter Meinl
Hans Rudolf Glatt
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Deutsches Institut fuer Ernaehrungsforschung Potsdam Rehbruecke
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Deutsches Institut fuer Ernaehrungsforschung Potsdam Rehbruecke
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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
    • C12N15/67—General methods for enhancing the expression

Definitions

  • the present invention relates to a method for changing the expression of polypeptides in recombinant expression systems, in which N-terminal codons in a gene to be expressed are replaced by a) synonymous and b) codons rare for the respective recombinant expression system.
  • the method can be carried out with a variety of different expression systems.
  • the invention further relates to a correspondingly mutated nucleic acid molecule in which N-terminal codons have been replaced by synonymous codons which are rare for the respective recombinant expression system, and to host cells and a method for expressing the mutated genes.
  • the amino acid sequence of a protein can be derived from the nucleotide sequence of the gene in question in units of three successive nucleobases (one codon).
  • mRNA messenger RNA
  • tRNA transfer RNA
  • the genetic code in living nature is (almost) universal, which means that all organisms use the same code.
  • the amino acids arginine, leucine and serine can each be coded by six, alanine, glycine, proline, threonine and valine by four each and isoleucine by three different base sequences.
  • the frequency analysis shows an unequal use of the principally equivalent codons depending on the species, with some codons occurring extremely rarely in the messenger RNAs, especially in the case of the amino acids mentioned above. Due to the rapid development in genetic engineering, many different approaches are now available for targeted changes in the codons of a gene.
  • One type of change concerns the synonymous base exchange, which alters the nucleotide sequence, but leaves the amino acid sequence unchanged. However, there are only a few studies on the consequences of such a base exchange. It is postulated that the frequency of the codons used reflects the frequency of the tRNA which recognizes them [1]. It has been shown that such rarely used codons do not occur in mRNA sequences that code for highly expressed proteins.
  • rare codons could be a regulatory mechanism that prevents the over-expression of these proteins [1].
  • expression of a protein in E. coli can be reduced by replacing synonymous codons with rare codons, in particular in a sequence of several rare codons in succession [2-4].
  • proteins are produced on an industrial scale in particular unicellular organisms and, among other things, in the production and preservation of food (e.g. dairy products, baked goods), in the production of complex chemicals and detergents, in agriculture and - last but not least - in the pharmaceutical sector Industry (eg insulin and other hormones, cytokines, antibiotics) are used.
  • Further examples are recombinant vaccines, microbial insecticides, technical enzymes (proteases, amylases, cellulases) and microbial polyesters.
  • US 4,945,051 describes a DNA segment which codes for a signal peptide. Using this signal sequence, recombinantly produced human lysozyme is exported into the culture medium and can thus be produced on a large scale.
  • US 5,082,767 describes a method for determining non-random codon pairing in an organism. The results are used to optimize codon pairing and to control translation efficiency.
  • US 5,436,391 describes a synthetic insecticidal gene in which the codon use has been adapted to Graminaceae and in particular rice.
  • No. 5,874,304 describes a “humanized” “green fluorescent protein” (GFP) which is adapted for high expression in mammalian cells, in particular in human cells. The codon usage is changed to a more suitable one for humans.
  • GFP green fluorescent protein
  • WO 99/14338 describes the codon adaptation of a LIP1 lipase from the yeast Candida rugosa to the expression in S. cerevisiae.
  • the object of the present invention is achieved by a method for changing the expression of polypeptides in recombinant expression systems, in which N-terminal codons in a gene to be expressed are replaced by a) synonymous and b) codons rare for the respective recombinant expression system.
  • the tRNAs compete for the amino acids, some of the rarer tRNAs may be activated differently than others. In addition, their stabilities could have a different regulatory effect at the start of translation at the N-terminus of the protein than at any other point on the protein. Therefore, the positioning of the codons plays a significant role in their effects.
  • the present invention relates to reverse substitution, i.e. rare codons are exchanged for more frequent ones.
  • N-terminus means the region between the start codon for methionine or F-Met (hereinafter referred to as codon “1”) to approximately amino acid 20 of the translated peptide.
  • codon “1” the start codon for methionine or F-Met
  • the areas to be modified can also be immediately connected to highly served leader, signal and / or translocation signals of the growing peptide chain lie, but this need not necessarily be the case.
  • a method according to the invention is preferred; in which at least one of the N-terminal codons 1 to 20 of the N-terminus of the gene to be expressed is replaced. It is further preferred that at least one of the N-terminal codons 1 to 10 of the N-terminus of the gene to be expressed is replaced.
  • Non-human animals or their tissues such as, for example, Drosophila, Caenorhabditis, sheep, cows, goats, rabbits, mice, rats, hamsters, zebrafish, monkeys and pigs, and organs of these animals,
  • Plants or their tissues such as, for example, arabidopsis, lupines, rice, potatoes, maize, oilseed rape, tomato, their leaves and callus cultures of the plants mentioned, and organs of the plants,
  • Mushrooms and their tissues such as mycorrhiza or tuber melanosporum,
  • prokaryotic individual cells such as, for example, Gram-positive or Gram-negative bacteria, such as, for example, Bacillus subtilis, Lactococcus, Escherichia coli, Salmonella typhimurium, Caulobacter, Pseudomonas or Streptomycetes,
  • Eukaryotic individual cells such as yeasts such as Saccharomyces, Hansenula and Pichia, insect cell lines and mammalian cell lines such as CHO cells, V79 cells, COS cells or human or monkey cell lines, and
  • any recombinant gene can be of interest as the gene to be expressed.
  • These genes are preferably selected from bacterial genes, eukaryotic cDNAs and viral genes.
  • the expression of genes which code for proteins of economic interest is particularly preferred.
  • the weak expression of problematic (poorly or only laboriously) to be expressed genes can be improved by means of the method according to the invention.
  • the further preferred genes of the method according to the invention are selected from the genes for hormones, cytokines, antibiotics, proteases, amylases, lipases, cellulases, insecticides, antibodies and fragments thereof, polymerases, restriction enzymes, enzymes which are involved in the synthesis of secondary metabolites in plants or fungi are involved and selective onsmarkern.
  • the genes for insulin, chitosan, chymosin, lysozyme, interferons, interleukins, growth hormones, erythropoietin, foreign matter-processing enzymes and transmembrane transporters are very particularly preferred.
  • Another aspect of the present invention relates to a method, wherein the gene to be expressed is selected from the genes for xenobiotic-eliminating enzymes, such as, for example, the cytochrome P-450, alcoholdehydrogenases, glutathione transferases, N-acetyltransferases, UDP-glucuronosyltransferases and sulfotransferases ( SULT).
  • the genes for xenobiotic-eliminating enzymes such as, for example, the cytochrome P-450, alcoholdehydrogenases, glutathione transferases, N-acetyltransferases, UDP-glucuronosyltransferases and sulfotransferases ( SULT).
  • this relates to a method which is characterized in that the expression system is Salmonella typhimurium LT2 and in the gene to be expressed at least one ⁇ -terminal codon for alanine by GCU or GCA, for arginine by AGA or AGG or CGA or CGG, for glutamine by CAA, for glycine by GGA or GGG, for isoleucine by AUA, for leucine by UUA or UUG or CUU or CUC or CUA, for lysine by AAG, for proline by CCU or CCC or CCA, for Serine is replaced by UCU or UCC or UCA or AGU, for threonine by ACU or ACA and for valine by GUA or GUG.
  • the expression system is Salmonella typhimurium LT2 and in the gene to be expressed at least one ⁇ -terminal codon for alanine by GCU or GCA, for arginine by AGA or AGG or CGA or CGG, for glutamine by
  • this relates to a method which is characterized in that the expression system is Escherichia coli Kl 2, and in the gene to be expressed at least one ⁇ -terminal codon for alanine by GCU, for arginine by AGA or AGG or CGA or CGG, for glutamate by GAG, for glycine by GGA or GGG, for isoleucine by AUA, for leucine by UUA or UUG or CUU or CUC or CUA, for lysine by AAG, for proline by CCU or CCC or CCA, for Serine is replaced by UCA, for threonine by ACU or ACA and for valine by GUA.
  • this relates to a method which is characterized in that the expression system is Lactococcus lactis, and in the gene to be expressed at least one ⁇ -terminal codon for Ala in by GCC or GCG, for arginine by AGG or CGC or CGG, for asparagine by AAC, for aspartate by GAC, for cysteine by UGC, for glutamine by CAG, for glutamate by GAG, for glycine by GGC or GGG, for histidine by CAC, for isoleucine by AUA or AUC, for leucine by CUC or CUG or CUA, for lysine by AAG, for phenylalanine UUC, for proline by CCC or CCG, for serine by UCG or UCC, for threonine by ACC or ACG, for tyrosine by UAC and for valine by GUC or GUG.
  • the expression system is Lactococcus lactis, and in the gene to be expressed at least one
  • this relates to a method which is characterized in that the expression system is Saccharomyces cerevisiae and in the gene to be expressed at least one N-terminal codon for alanine by GCG, for arginine by CGC or CGA or CGG, for glutamine by CAG, for glutamate by GAG, for glycine by GGC or GGG, for leucine by CUU or CUC or CUG or CUA, for proline by CCC or CCG, for serine by UCG or AGC, for threonine by ACG and for valine GUG is replaced.
  • the expression system is Saccharomyces cerevisiae and in the gene to be expressed at least one N-terminal codon for alanine by GCG, for arginine by CGC or CGA or CGG, for glutamine by CAG, for glutamate by GAG, for glycine by GGC or GGG, for leucine by CUU or CUC or CUG or CUA, for proline
  • this relates to a method which is characterized in that the expression system is derived from Homo sapiens and in the gene to be expressed at least one N-terminal codon for alanine by GCG, for arginine by CGU or CGA, for glutamine by CAA, for glutamate by GAG, for glycine by GGU, for isoleucine by AUA, for leucine by UUA or UUG or CUU or CUA, for proline by CCG, for serine by UCG, for threonine by ACG and for valine by GUU or GUA is replaced.
  • the expression system is derived from Homo sapiens and in the gene to be expressed at least one N-terminal codon for alanine by GCG, for arginine by CGU or CGA, for glutamine by CAA, for glutamate by GAG, for glycine by GGU, for isoleucine by AUA, for leucine by UUA or UUG or CUU or CUA,
  • this relates to a method which is characterized in that the expression system is Critelus griseus or is derived therefrom, and in the gene to be expressed at least one N-terminal codon for alanine by GCG, for arginine by CGU or CGA, for glutamine by CAA, for glutamate by GAG, for glycine by GGU, for isoleucine by AUA, for leucine by UUA or UUG or CUU or CUA, for proline by CCG, for serine by UCA or UCG, for threonine by ACG and for Valin is replaced by GUU or GUA.
  • the expression system is Critelus griseus or is derived therefrom, and in the gene to be expressed at least one N-terminal codon for alanine by GCG, for arginine by CGU or CGA, for glutamine by CAA, for glutamate by GAG, for glycine by GGU, for isoleucine by AUA, for leucine by
  • this relates to a method which is characterized in that the expression system is or is derived from Oryza sativa, and in the gene to be expressed at least one N-terminal codon for alanine by GCA, for arginine by CGU or CGA, for glycine by GGU or GGA, for isoleucine by AUA, for leucine by UUA or CUA, for lysine by AAA, for serine by AGU and for valine by GUA.
  • this relates to a method which is characterized in that the expression system is Arabidopsis thaliana or is derived therefrom, and in the gene to be expressed at least one N-terminal codon for alanine by GCC or GCG, for arginine by CGC or CGA or CGG, for aspartate by GAC, for glycine by GGC or GGG, for isoleucine by AUA, for leucine by UUA or CUA or CUG, for proline by CCC or CCG, for serine by UCC or UCG or AGC, for threonine is replaced by ACG and for valine by GUC or GUA.
  • the expression system is Arabidopsis thaliana or is derived therefrom, and in the gene to be expressed at least one N-terminal codon for alanine by GCC or GCG, for arginine by CGC or CGA or CGG, for aspartate by GAC, for glycine by GGC or GGG, for isoleucine
  • the present invention relates to reverse substitution, i.e. rare codons are exchanged for more frequent ones.
  • the frequencies of the individual codons are different for each organism.
  • the organisms shown above are therefore only a selection of sample organisms. How the codon frequency is to be determined and how the sequence to be expressed is then to be adapted to the respective host organism can be found in the abovementioned examples and the following table of the codon frequencies and the literature available in the prior art on codon adaptations and can be adapted without difficulty to all other organisms and expression systems which are not yet listed in Table 1 below.
  • the codon frequency can also be adapted accordingly to the enzymes and tRNAs present for in vitro expression. For this purpose, these systems can also be modified accordingly compared to the commercially available systems.
  • / c (a) frequency with which codon c is used for coding amino acid a;
  • the way in which the nucleotide exchanges are introduced into N-terminal codons to be modified can be carried out by any suitable means available in the prior art.
  • suitable means available in the prior art.
  • a conventional mutagenesis method such as, for example, PCR mutagenesis (for example using mutated primers or the errors introduced by the polymerase), site-specific mutagenesis (“site-directed mutagenesis”), ligation mutagenesis , chemical mutagenesis by means of suitable chemicals or mutagenesis by homologous recombination.
  • a further aspect of the present invention then relates to a nucleic acid molecule in which N-terminal codons have been replaced by synonymous codons which are rare for the respective recombinant expression system and which is produced by means of one of the methods according to the invention mentioned above.
  • a further aspect of the present invention relates to a DNA or RNA vector molecule which comprises at least one or more nucleic acid molecule (s) as mentioned above and which can be expressed in a suitable expression system. This expression is carried out by the inserted mutations in a controlled or adapted manner.
  • Another aspect of the present invention relates to a host cell or a host organism that expresses a DNA or RNA vector molecule according to the present invention. All expression systems mentioned above can be used as organisms.
  • Pro or eukaryotic single cells such as, for example, Gram-positive or Gram-negative bacteria, yeasts, insect cell lines and mammalian cell lines, such as, for example, CHO cells, V79 cells, COS cells, human cell lines or monkey cell lines, are particularly preferred.
  • a further aspect of the present invention then relates to a method for expressing a recombinant polypeptide in an expression system, comprising the steps of a) providing the gene for the polypeptide to be recombinantly expressed, b) providing a suitable expression system, c) performing one method according to the invention as mentioned above, d) introducing the mutated gene from step c) into the suitable expression system, and e) expression of the gene and obtaining the polypeptide to be expressed recombinantly from the expression system.
  • the type of introduction into the expression system in step d) depends on the particular expression construct that is to be introduced into the system.
  • Types of transformation or transfection are known to those skilled in the art and include chemical and physical transfer methods such as, but not limited to, electroporation, particle transfer, liposome transfer, infection, conjugation, coprecipitation method or DMSO-mediated transformation.
  • the receipt and expression of the polypeptide to be recombinantly expressed from the expression system in step e) also depends, inter alia, on the type of expression system and the polypeptide to be expressed. In some cases this can also be cleaned from the culture medium.
  • the expressed protein can be contained in body fluids such as blood and urine or in animal products such as milk. A large number of corresponding cleaning methods are known to the person skilled in the art. These can easily be adjusted accordingly.
  • a last aspect of the present invention then relates to the recombinant polypeptide which is obtained by a process according to the invention.
  • FIG. 2 shows an immunoblot of the cytosol of the Salmonella typhimurium strains TA1538.
  • FIG. 3 shows an immunoblot of the cytosol of the Escherichia coli strains XL1-1A2 * 1 (* 1),
  • FIG. 4 an immunoblot of the cytosol of the E. coli strains XLl-2Blb (lb) (XL1-
  • SEQ ID No. 1 shows forward primer (FI) 5'-GAG CTC AGG ACC ATG GAG CTG ATC,
  • SEQ ID No. 2 shows the reverse primer (R1) 5 * -ACT CTC TCT AGA GTG ACC CCA GGA
  • SEQ ID No. Figure 3 shows forward primer (F4): 5'-AACAGACC ATG GAG CTA ATA CAA GAC
  • SEQ ID No. Figure 4 shows forward primer (F5): 5'-AACAGACC ATG GAG CTA ATA CAA GAC
  • SEQ ID No. Figure 5 shows forward primer (F2): 5'-GAAGAAGAACCCTATGACCAAC.
  • SEQ ID No. 6 shows forward primer (F3) 5'-CTC AGG ACC ATG GAG CTG ATC CAG
  • SEQ ID No. 7 shows forward primer (F6): 5'-TCCCTAGTCGACGCCATGGCGTCTCCC,
  • SEQ ID No. 8 shows reverse primer (R2): 5'-
  • SEQ ID No. 9 shows forward primer (F7): 5'-GAC GGA CCA GCA GAG CCA CAA ATA
  • SULT1A2cDNA was isolated from human liver samples using the reverse transcriptase (RT) polymerase chain reaction (PCR).
  • Total RNA was under isolated using the RNeasy mini kit from Qiagen (Hilden, Germany). 2 ⁇ g of total RNA were subjected to an RT with Superscript TM II (Lifetechnologies, Rockville, MD, USA) using an 18-mer oligo-DT primer (Bio TEZ, Berlin, Germany) at 42 ° C. for one hour , 2 ⁇ l of the RT reaction product were amplified for SULT1 A cDNA in subsequent PCR using two units of Kombipol TM -DNA polymerase mix (Invitec, Berlin, Germany) in a final volume of 50 ⁇ l according to the recommendations of Manufacturer uses.
  • RT reverse transcriptase
  • the forward primer (FI) 5'-GAG CTC AGG ACC ATG GAG CTG ATC (SEQ ID No. 1) introduced an Nco I site at the position of the translation start codon of SULTA2.
  • the reverse primer (R1) 5'-ACT CTC TCT AGA GTG ACC CCA GGA GC (SEQ ID No. 2) introduced a 3'-Xba I restriction site in the 3 'flanking region.
  • the reaction conditions for the PCR were: 5 minutes at 95 ° C for denaturation, 1 minute at 94 ° C for the later denaturation steps, 1 minute at 60 ° C for annealing and 1 minute at 72 ° C for extension (30 cycles); and a final extension step at 72 ° C for 10 minutes.
  • this primer would amplify all SULT1 A cDNAs that are present in the sample. Therefore, a Barn HI (MBI-Fermentas, Wilnius, Lithuania) restriction digest was carried out followed by 1% agaraoe gel electrophoresis in order to specifically digest and resolve the SULTA1 cDNA. The remaining 900 bp full length SULT cDNA was extracted from the gel using the Qiaex II gel extraction kit (Qiagen) and then used as a template for a second PCR under the same conditions to enrich the PCR product for SULT1A2.
  • Qiaex II gel extraction kit Qiagen
  • SULT1A2 * 1 cDNA was constructed by site-directed in vitro mutagenesis of SULT1A2 * 2 cDNA in two successive PCR reactions.
  • the codon for Thr was mutated to an Asn codon (AAC) using the internal forward primer (F2): 5'-GAAGAAGAACCCTATGACCAAC (SEQ ID No. 5).
  • the forward primer (F3) 5'-CTCAGGACCATGGAGCTGATCCAGGACATCTCT (SEQ ID No. 6) in the second PCR reaction was designed so that codon 7 encoded Ile (ATC) instead Thr (ACC) and that a silent C to T mutation between SULT1A2 * 2 and SULT1A2 * 1, which was described in the sequence database, was also introduced.
  • the resulting long length product was cloned into the modified prokaryotic expression vector pKK233-2 using the 5'-Nco I and the 3'-Xba / restriction site. Sequencing confirmed that the subcloned cD ⁇ A was identical to that reported in the database for SULT1A2 * 1 (HAST4v) (Genbank, accession number U28169).
  • the sequence of the forward primer F5 5'-AACAGACC ATG GAG CTA ATA CAA GAC ACA TCA AGG CCG CCA C was used and the PCR was carried out with the reverse primer R1 (SEQ ID ⁇ o . 2) and the SULT1 A2 * 2 cD ⁇ A template. In both cases the PCR conditions were the same as for the previous constructs.
  • the vector pKK233-2 had proven to be suitable for the constructive expression of SULT in E. coli and S. typhimurium.
  • the polylinker region of this vector only exists from three restriction sites, Nco I (at the 5 'end), Pst I and Hind III. This caused a problem because SULT1A2 cDNA has three internal Pst I sites and one Hind III restriction site.
  • an Nco VPvu I fragment of pKK233-2 was replaced by the corresponding fragment of the prokaryotic expression vector pTRC99A (Pharmacia), which is a derivative of pKK233-2 and which pUC18 polylinker site exchanged.
  • the sequence of the modified pKK233-2 vector is identical to pKK233-2, except for the polylinker site of the newly inserted fragment from pTRC99A.
  • SULTBlb cDNA was isolated from human placenta samples using RT-PCR.
  • the reverse transcription was carried out analogously to the SULTlA2 isolation.
  • the forward primer F6 5'-TCC CTA GTC GAC GCC ATG GCG TCT CCC (SEQ ID No. 7)
  • the reverse primer R2 5'-ACT GAC AAG CTT ATT ATG AGG GTC GTG G (SEQ ID No . 8) which insert an Nco I at the 5 'end of the amplicate and an Hin dlll restriction site at the 3' end.
  • the number of PCR cycles was increased to 40, otherwise the PCR was carried out as described for SULT1A2.
  • the PCR resulted in a homogeneous amplification, which was ligated via the introduced restriction sites into the p-KK233-2 vector, which had also been digested with Nco I / Hin dlll, and which could be transfected into E. coli.
  • An E. coli clone could be isolated in which the cDNA inserted into the pKK233-2 Velctor matched the SULT2Blb reference sequence (GenBank, accession number U92315).
  • the plasmid was then transferred into S. typhimurium TA1538 analogously to the SULT1A2 plasmids.
  • the wild-type SULT2Blb cDNA was only weakly expressed in S. typhimurium. 6-20 times more protein compared to other SULT had to be used for the Western blot to get a clear signal.
  • a few codons after the translation start codon were exchanged for those which are rarely used by S. typhimurium, analogously to the method described.
  • the forward primer F7 5'-GAC GGA CCA GCA GAG CCA CAA ATA CCA GGA CTA TGG GAC (SEQ ID No. 9), together with R2 (SEQ ID No.
  • This artificial cDNA which is synonymous with SULT2Blb * l, was named SULT2Blb * lX.
  • Primer F7 did not contain the Nco I site and the translation start codon to limit the length.
  • the vector was restricted with Nco I.
  • the resulting overhang of the opposite strand was filled in with DNA polymerase I (Klenow fragment), so that a smooth end with the sequence ATG was formed.
  • the SULT2Blb * lX cDNA was then cloned into the vector with its smooth 5 'end and the Hin dlll restricted 3' end.
  • Cytosolic proteins (1-30 ug per lane) were resolved by SDS-polyacrylamide (110 mg / ml gel electrophoresis) according to the Lämmli method. After electrophoresis, the Hyobond ECL membrane proteins (Amersham Pharmacia Biotech, Little Chalfont, UK) were transferred. Two immune sera were used to detect SULT1 A2. An immune serum obtained in sheep against purified human SULT1 A3 protein recognized all human SULTIA proteins. The second immune serum was obtained in rabbits against a peptide (15 amino acids) contained in SULT1A1 and SULT1A2, but not in SULT1A3. Therefore, the serum only recognizes the first two forms.
  • SULT2Blb For the detection of SULT2Blb, an immune serum obtained in rabbits against the splice variant SULT2Bla was used, which also recognizes SULT2Blb, but no other human SULT.
  • the bands recognized by the antibody were analyzed using stronger chemiluminescence (Amersham) visualized with the help of the Fuji LAS-lOOO imaging system (Raytests, Straubenhardt, Germany).

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Abstract

L'invention concerne un procédé pour modifier l'expression de polypeptides dans des systèmes d'expression recombinants, selon lequel, dans un gène à exprimer, des codons de N-terminale sont remplacés par a) des synonymes et b) des codons rares pour le système d'expression recombinant correspondant. Ce procédé peut être réalisé avec une pluralité de systèmes d'expression différents. La présente invention porte également sur une molécule d'acide nucléique mutée en conséquence, dans laquelle des codons de N-terminale sont remplacés par des synonymes et par des codons rares pour le système d'expression recombinant correspondant. Ladite invention concerne aussi des cellules hôtes et un procédé pour l'expression des gènes mutés.
PCT/EP2003/013410 2002-11-29 2003-11-28 Procede pour modifier l'expression de polypeptides dans des systemes d'expression recombinants Ceased WO2004050878A1 (fr)

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DE2002155852 DE10255852B4 (de) 2002-11-29 2002-11-29 Verfahren zur Expressionsveränderung von Polypeptiden in rekombinanten Expressionssystemen
DE10255852.3 2002-11-29

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2374880A4 (fr) * 2008-12-05 2013-05-22 Japan Agency Marine Earth Sci Procédé d'inhibition de la prolifération de cellules

Citations (2)

* Cited by examiner, † Cited by third party
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US9492568B2 (en) * 2008-12-05 2016-11-15 Japan Agency For Marine-Earth Science And Technology Method for suppressing cell growth

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