WO2004085649A1 - Facteur prfb (facteur de liberation de chaine peptidique b) issu de bacillus licheniformis et utilisation de celui-ci pour l'augmentation du taux de formation de proteines - Google Patents

Facteur prfb (facteur de liberation de chaine peptidique b) issu de bacillus licheniformis et utilisation de celui-ci pour l'augmentation du taux de formation de proteines Download PDF

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WO2004085649A1
WO2004085649A1 PCT/EP2004/001950 EP2004001950W WO2004085649A1 WO 2004085649 A1 WO2004085649 A1 WO 2004085649A1 EP 2004001950 W EP2004001950 W EP 2004001950W WO 2004085649 A1 WO2004085649 A1 WO 2004085649A1
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prfb
factor
protein
seq
particularly preferably
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PCT/EP2004/001950
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German (de)
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Maren Hintz
Roland Freudl
Jörg FEESCHE
Roland Breves
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Henkel Kommanditgesellschaft Auf Aktien
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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
    • 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
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present application relates to the factor PrfB (peptide chain release factor B; RF-2) from Bacillus licheniformis and to all factors PrfB which are at least 93% identical at the amino acid level and at least 80% identical at the DNA level. It also relates to the additional provision of the PrfB factor and / or a PrfB factor whose activity has been improved in order to increase the protein formation rate in processes for protein production by gram-positive or gram-negative bacteria.
  • PrfB peptide chain release factor B
  • genes for the proteins of interest are introduced into host cells and transcribed, translated and, if necessary, secreted by the membranes in question into the periplasm or the surrounding medium.
  • Host cells established especially for this purpose are gram-negative bacteria, such as, for example, Escherichia coli or Klebsiella, or gram-positive bacteria, such as, for example, species of the genera Staphylococcus or Bacillus.
  • bacterial systems for protein production are selected which are established and inexpensive to ferment, which promise a high product formation rate and which ensure correct folding, modification, etc. of the protein to be produced. The latter is all the more probable as the relationship with the organism originally producing the protein of interest increases. Suitable systems are determined experimentally in individual cases. A high product formation rate is particularly desirable for economic reasons.
  • the translocation is naturally preceded by protein synthesis, which is also indicated in FIG. 5.
  • the The resulting peptide chain is kept in an unfolded state by cytoplasmic proteins with chaperone function and transported to the membrane. Then the transport of the peptide through the membrane is catalyzed with ATP consumption (Mitchell, O, Oliver, D. (1993): "Two distinct ATP-binding domains are needed to promote protein export by Escherichia coli SecA ATPase", Mol. Microbiol ., Volume 10 (3), pages 483-497), where SecA acts as an energy-supplying component (ATPase).
  • the signal peptide After overcoming the membrane, the signal peptide is split off by the activity of a signal peptidase and the extracellular protein is thus detached from the membrane.
  • the exoproteins In the case of gram-positive bacteria, the exoproteins are discharged directly into the surrounding medium. In Gram-negative bacteria, the proteins are then usually in the periplasm and further modifications are required to achieve their release into the surrounding medium.
  • the protein formation rate can be achieved by overexpression of the factor PrsA involved in the translocation.
  • the two applications WO 99/04406 A1 and WO 99/04407 A1 teach that protein production by gram-positive bacteria can be increased by an increased expression of the transmembrane proteins SecG or SecDF.
  • the application WO 01/81597 A1 relates to processes for the production of recombinant proteins by gram-negative bacteria, which are characterized in that the products are released into the surrounding medium. This is achieved in that, at the same time as the transgene-regulating system, a second system becomes active, which partially opens the outer membrane of the bacteria.
  • a second system becomes active, which partially opens the outer membrane of the bacteria.
  • the factor PrfB (peptide chain release factor B; also RF2) is known as part of the apparatus for protein synthesis from the prior art, both in gram-positive and in gram-negative bacteria. In connection with translation, he is responsible in particular for the detachment of the finished translated proteins from the ribosome, that is to say for the termination of the translation. This is indicated by an arrow in FIG. 5. However, the connection with the translocation also shown there and explained above is only indirect, namely that the prfB gene is expressed in many bacteria simultaneously with a gene for a translocation protein (cf. Ex. 1 and FIG. 3). According to the publication by Herbort et al.
  • subtilis are compared and a five-domain structure is proposed for them Interaction of this factor with the mitochondrion.
  • Domain V of the respective factor should have the task of to interact with the stop codon of the mRNA, and the domain I reaching approximately to amino acid 110 of the PrfB from B. subtilis is said to bind to the ribosome, at the point of contact of the two ribosomal subunits, near the L7 / L12 finger of the mediate large subunit.
  • the PrfB factor has therefore already been isolated and sequenced from various microorganisms.
  • sequences in question are usually stored in generally accessible databases, for example at the National Center for Biotechnlogy Information (NCBI), National Library of Medicine, Building 38A, Bethesda, MD 20894, USA; accessible via http://www.ncbi.nlm.nih.gov/.
  • NCBI National Center for Biotechnlogy Information
  • those from E. coli and Bacillus subtilis have the registration numbers Q8XD56 and NP_391409.
  • the factor PrfB has not yet been described from B. licheniformis.
  • PrfB PrfB
  • B. licheniformis No commercial applicability has yet been proposed for PrfB, especially not for the molecules from B. licheniformis or highly homologous ones.
  • the provision of this factor in excess to increase the protein synthesis rate and thus the yield of protein produced overall has not yet been described in the prior art. It would come into consideration for the fermentative production of proteins and processes based thereon for protein production.
  • the object was to establish, in addition to the systems described in the prior art, a further system by which the processes for protein production by fermentation of bacteria can be further improved in terms of an increased yield.
  • the solution to this problem according to the invention shows two fundamental aspects. On the one hand, it is solved by providing a factor PrfB (peptide chain release factor B) with an amino acid sequence that corresponds to that in SEQ ID NO. 2 given amino acid sequence has a certain homology.
  • the other basic aspect consists in the provision of methods for protein production by fermentation of bacteria, which are generally characterized in that the factor PrfB (peptide chain release factor B) is present in the cells producing the protein with a higher activity than naturally in these cells , This second aspect brings this factor to an important commercial application. Because, as was surprisingly found in the model system B. licheniformis, an increased PrfB activity leads overall to an increase in the yield of protein production (example 3).
  • the cited first aspect of the present invention relates to a factor PrfB (peptide chain release factor B) with an amino acid sequence that corresponds to that in SEQ ID NO. 2 amino acid sequence indicated has a homology of at least 93% identity.
  • Example 1 of the present application shows how, based on a known p / ⁇ sequence, that of an unknown PrfB can be found.
  • that from B. licheniformis was identified on the basis of the sequence known for B. subtilis.
  • Both the amino acid and the protein sequence of the PrfB from B. subtilis are stored in the known databases; they are also listed under SEQ ID NO. Specify 9 and 10.
  • the DNA sequence can be used as a probe to find the homologs in question in other organisms.
  • the other sequences specified in the sequence listing can also be used for the same purpose. It is thus possible for the person skilled in the art to find further PrfB using routine methods and in particular based on Example 1.
  • a regulatory specialty of PrfB is that there is a reading frame shift in the associated gene.
  • E. coli this is, for example, in the publication "Identification of the mutations in the prfB gene of Escherichia coli K12, which confer UGA suppressor activity "by Wu, ED, Inokuchi, H. and Ozeki, H., in Jpn. J. Genet. (1990), Volume 65 (3), pages 115 to 119.
  • a correct one B. licheniformis PrfB is obtained only by skipping the T in position 73 during the transcription and only continuing from the following position, which regulates the expression of this factor, as is also noted in the sequence listing of the present application. This applies both to the wild type sequence from B. licheniformis DSM 13 (SEQ ID NO. 1 and 2) described in Example 1 and to the mutant B. licheniformis E described in Example 2 (SEQ ID NO. 3 and 4).
  • B. halodurans are in SEQ ID NO. 5 and 6, but also, for example, in the GenBank database (National Center for Biotechnology Information NCBI, National Institutes of Health, Bethesda, MD, USA; http://www3.ncbi.nlm.nih.gov) under the numbers NC_002570 and E48100 specified. There is no reading frame shift here. For this, this gene or protein has the special feature that the initiation codon TTG is translated with the amino acid methionine.
  • this gene codes prfB for a factor which, as stated in the introduction, takes on the physiologically important function of catalyzing the termination of translation in the course of protein biosynthesis. In this sense, it is a peptide chain release factor B.
  • homologs with at least 93% identity at the amino acid level are claimed according to the invention.
  • this factor can be used commercially in particular in that it can serve to increase the yield in protein production by culture of microorganisms.
  • those PrfB are particularly preferred which have an advantageous effect in terms of increasing the yield in protein production by culture of microorganisms.
  • These include in particular those variants of wild-type enzymes which have been improved with Mutagense with regard to this possible use.
  • PrfB with an amino acid sequence that corresponds to that in SEQ ID NO. 2 amino acid sequence increasingly preferred a homology of 94%, 94.5% 95%, 95.5%, 96%, 96.5%, 97%, 97.5% 98%, 98.5%, 99%, 99 , 5% identity and particularly preferably 100% identity.
  • the PrfB from ß. licheniformis DSM 13 has been investigated in Examples 2 and 3 B. licheniformis E. As stated there and also shown in FIGS. 1 and 2, it is characterized by numerous mutations, of which only two, however, penetrate to the amino acid level. These are the two positions that are in homologous positions 71 and 100 of the mature protein according to SEQ ID NO. 2 correspond and there the amino acids asparagine (N) or serine (S). Compared to the wild-type molecule, the PrfB is from ß. licheniformis E is a double variant S71N / N100S.
  • this mutation has an advantageous effect on the protein formation or at least protein secretionates. Without wishing to be bound by this theory, one might assume that this effect is due to improved intramolecular interactions within this factor. Because of the fact that PrfB is technically important in accordance with the invention as a factor that improves protein production, such variants are particularly preferred.
  • the factor PrfB is not actually obtained from ⁇ . licheniformis DSM 13 but that of the associated nucleic acid. This is also in SEQ ID NO. 1 and compared to that of prfB from ß. licheniformis E indicated in Figure 2. Since this has not been previously described and the next similar gene, namely the prfB from ß. subtilis has a similarity of 78.5% identity at the DNA level, the protection range extends to correspondingly more similar nucleic acids.
  • nucleic acid with a nucleotide sequence which corresponds to that in SEQ ID NO. 1 nucleotide sequence increasingly preferred a homology of 82.5%, 83%, 84%, 85%, 86%, 87%, 87.5%, 88%, 89%, 90%, 91%, 92%, 92, 5%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 99%, 99.5% identity and particularly preferably 100% identity.
  • nucleic acid in question has one, preferably both, positions which are in homologous positions 71 and 100 of the mature protein according to SEQ ID NO. 2 correspond, encoded for the amino acids asparagine (N) or serine (S).
  • the protection also applies to the associated nucleic acids. This is because the present invention is realized in particular via these if, for example, the relevant factor is to be provided in an increased activity in a specific cell.
  • embodiments of the present invention are nucleic acids coding for a factor PrfB with a nucleotide sequence which corresponds to that in SEQ ID NO. 1 indicated nucleotide sequence have a homology of at least 80% identity.
  • nucleic acids coding for a factor PrfB are increasingly preferred, which correspond to those in SEQ ID NO. 1 indicated nucleotide sequence a homology of 82.5%, 83%, 84%, 85%, 86%, 87%, 87.5%, 88%, 89%, 90%, 91%, 92%, 92.5% , 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 99%, 99.5% identity and particularly preferably 100% identity.
  • nucleic acids coding for a factor PrfB which are characterized in that they have one, preferably both, positions which are in homologous positions 71 and 100 of the mature protein according to SEQ ID NO. 2 correspond, for which amino acids encode asparagine (N) or serine (S).
  • the present invention is accomplished by providing the vectors concerned. Because the genes in question can be characterized, cultivated and introduced into host cells. Accordingly, vectors which contain one of the nucleic acids described above, in particular those which encode PrfB for one of the factors described above.
  • Preferred such vectors are characterized in that they are cloning vectors and / or expression vectors, preferably those that can be kept stable in the derived bacterial strains after transformation.
  • the cloning vectors in particular are used for characterization, for example by sequencing or permanent storage.
  • Expressin vectors implement the present invention by causing the transformed cells to produce the protein in question. In this way, they are effective according to the invention. It is particularly advantageous if in this way permanently transformed strains are obtained which, for example, can be developed into a group of derived, related strains by additionally transforming them with other genes for proteins of interest intended for production. Replication origins and resistance markers present on the plasmids in particular allow the plasmids in question to be kept stable in the cells.
  • the prB gene is under the control of an inducible promoter. In this way, the rate of protein formation can be increased from outside at a certain point in time.
  • the invention is also implemented by cells which have been obtained by transformation with one of the nucleic acids described or which are derived from such a cell, in particular by transformation with one of the vectors described.
  • this first aspect of the invention is to mention methods for producing a factor PrfB described above. Because of such processes you get this factor in its pure form. In accordance with what has been said above, this takes place molecular-biologically using one of the nucleic acids described, particularly preferably using one of the vectors described and very particularly preferably using a previously described cell containing or expressing this gene. Because these steps are usually used to obtain the factor in pure form.
  • the second aspect of the present invention consists in methods for protein production by culturing bacteria, which are characterized in that the factor PrfB (peptide chain release factor B) has a higher activity in the cells producing the protein than naturally in these Cells.
  • PrfB peptide chain release factor B
  • Methods for protein production by culturing bacteria are known per se. In principle, all protein production processes can be further improved in this way. This applies in particular to processes which are based on cultivation in liquid media and, in particular, on fermentation.
  • the implementation of the present invention is not restricted solely to the factors PrfB provided with the present application.
  • all PrfB that function in the cells intended for protein production can be used. This seems all the more promising the closer the PrfB in question is to the PrfB present endogenously in these cells.
  • a preferred method for protein production by culturing bacteria is characterized in that the higher PrfB activity is due to additional expression of the gene prfB coding for this factor PrfB.
  • Example 3 This is illustrated by Example 3, where, in addition to the endogenous prfB, a prfB is introduced on a plasmid which carries a constitutive promoter for this prfB.
  • the protein formation rate is increased compared to the comparison strain transformed with the empty vector.
  • a particularly preferred method is characterized in that the additional expression of the factor PrfB is due to additional copies of the gene prfB in the genome of the bacteria in question, preferably because of their stable establishment in the derived bacterial strains after previous transformation.
  • a stable establishment can be achieved, for example, by means of suitable selection markers located on the vectors with the pfß gene or by integrating the prfB into the bacterial chromosome. This would eliminate the selection, also made in Example 3, of an externally added substance.
  • Example 3 suggests that it makes sense to do this in ß. licheniformis DSM endogenous gene prfB against the point mutated gene from ß. licheniformis E. This can be done, for example, by homologous recombination.
  • a correspondingly preferred method according to the invention is thus characterized in that in the cells producing the protein the endogenous factor PrfB has been replaced by a PrfB with higher activity, preferably by replacing at least one endogenously present p / fß gene by the one for the PrfB with higher P / fß gene encoding activity.
  • the elements provided with the present application are used here and in all other previously described methods for protein production by culturing bacteria. Because, as shown in the examples in particular, these are useful tools for this.
  • Such methods are characterized in that the additional PrfB activity by a factor of PrfB according to the invention from ß. licheniformis related factor is achieved, preferably with the aid of a nucleic acid carried out in this connection, particularly preferably with the aid of a corresponding vector and very particularly preferably by means of a correspondingly transformed cell.
  • This cell is therefore the producer of a protein of interest, which additionally provides a PrfB according to the invention and is therefore improved in terms of its product formation rate.
  • a preferred method according to the invention is characterized in that the cells producing the protein are gram-positive bacteria, preferably of the genera Staphylococcus, Corynebacteria or Bacillus, especially of the species Staphylococcus carnosus, Corynebacterium glutamicum, Bacillus subtilis, B. licheniformis, B. amyloliquefaciens, B. globigii or ß. lentus, and especially around strains of ß. licheniformis or ß. amyloliquefaciens.
  • the cells producing the protein are gram-positive bacteria, preferably of the genera Staphylococcus, Corynebacteria or Bacillus, especially of the species Staphylococcus carnosus, Corynebacterium glutamicum, Bacillus subtilis, B. licheniformis, B. amyloliquefaciens, B. globigii or ß. lentus, and especially around strains of ß. lichen
  • an increase in performance according to the invention can be expected in particular if the factor PrfB which improves protein synthesis works well with the relevant protein synthesis machinery. There is a high probability of this if the PrfB originally comes from a related organism. This applies to the wild-type sequences of the PrfB provided and those PrfB which, based on these wild-type sequences, have been improved with regard to their performance increase in protein synthesis, in particular via point mutagenesis.
  • Such a method for gram-positive microorganisms is thus characterized in that the additional PrfB activity is achieved by a factor PrfB, the wild-type sequence of which comes from a gram-positive bacterium.
  • Such a method is preferred if it is characterized in that the additional PrfB activity is achieved by a factor PrfB which comes from the same genus as the gram-positive bacteria producing the protein, preferably from the same species, particularly preferably from the same strain.
  • PrfB is used which is based on the wild-type sequence. licheniformis, B. halodurans or ß. subtilis, preferably a wild-type sequence according to SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 10, particularly preferably encoded by a sequence which differs from a wild-type sequence according to SEQ ID NO. 3, SEQ ID NO. 5 or SEQ ID NO. 9 derives.
  • the cells producing the protein are Gram-negative bacteria, preferably of the genera E. coli or Klebsieila, in particular strains of Escherichia coli K12, Escherichia coli B or Klebsieila planticola, and especially derivatives of the strains Escherichia coli BL21 (DE3), E. coli RV308, E coli DH5 ⁇ , E. coli JM109, E. coli XL-1 or Klebsieila planticola (Rf).
  • E. coli or Klebsieila preferably of the genera E. coli or Klebsieila, in particular strains of Escherichia coli K12, Escherichia coli B or Klebsieila planticola, and especially derivatives of the strains Escherichia coli BL21 (DE3), E. coli RV308, E coli DH5 ⁇ , E. coli JM109, E. coli XL-1 or Klebsieila planticola (Rf).
  • methods based on these organisms are characterized in that the additional PrfB activity is achieved by a factor PrfB, the wild-type sequence of which comes from a gram-negative bacterium.
  • those methods are further preferred which are characterized in that the additional PrfB activity is achieved by a factor PrfB which comes from the same genus as the gram-negative bacteria producing the protein, preferably from the same species, particularly preferably from the same strain.
  • the present application also provides a concrete starting point for this, which is accordingly preferred, namely the p / f ⁇ sequence from the gram-negative model organism E. coli.
  • methods of this type are preferred which are characterized in that a PrfB is used which is derived from the wild-type sequence from Escherichia coli, preferably from the wild-type sequence according to SEQ ID NO. 8, particularly preferably encoded by a sequence which differs from a wild-type sequence according to SEQ ID NO. 7 derives.
  • PrfB performance-improved variants of PrfB are of particular interest according to the invention.
  • the PrfB was made from ß in Example 2.
  • licheniformis which has the exchanges S71N and N100S compared to the wild type.
  • Preferred protein production processes according to the invention are thus characterized in that an activity-improved variant of a factor PrfB is used, preferably one which is in one, particularly preferably both, positions in positions 71 and 100 of the mature protein according to SEQ ID NO. 2 correspond to the amino acids asparagine (N) or serine (S).
  • the gene coding for the protein produced is naturally present in the cells producing the protein, preferably this protein is naturally formed by these cells.
  • transgenes that is to say those which have been introduced into suitable host cells and are expressed by them. Because this enables strains optimized for production to be used for various purposes and the known molecular biological methods can be used to produce optimized proteins that do not occur in nature in this form.
  • the gene coding for the protein produced has been introduced into the progenitor cells of the bacteria used for the production by transformation, preferably via an expression vector, particularly preferably because of its stable establishment in the derived bacterial strains. Two examples are shown in Example 3.
  • Example 3 the protein formation rate can be determined by measuring the activity in question in the supernatant.
  • an alternative that is possible according to the invention also consists in disrupting the relevant protein-producing cells after the actual production and thereby obtaining the product.
  • preferred methods are characterized in that the protein produced is secreted.
  • protein production processes which are characterized in that the protein produced is a non-enzyme, preferably a pharmacologically relevant protein, particularly preferably insulin or calcitonin.
  • enzymes are also of great technical importance, for example for biotransformation, i.e. chemical synthesis using enzymatic catalysts or as effective components in detergents and cleaning agents.
  • those methods are also claimed which are characterized in that the protein produced is an enzyme, preferably a hydrolytic enzyme or an oxidoreductase, particularly preferably a protease, amylase, hemicellulase, cellulase, lipase, cutinase , Oxidase, peroxidase or laccase.
  • the representatives that are preferably produced for use in detergents and cleaning agents are listed below.
  • proteases those of the subtilisin type are preferred, for example the alkaline protease from Bacillus lentus.
  • the protease from Bacillus lentus DSM 5483 (WO 91/02792 A1) is derived from the variants listed under the name BLAP ® , which are described in particular in WO 92/21760 A1, WO 95/23221 A1, WO 02/088340 A2 and WO 03 / 038082 A2.
  • gibsonii go from the patent applications WO 03/054185 A1, WO 03/056017 A2, WO 03/055974 A2 and. WO 03/054184 A1.
  • amylases which can be produced according to the invention are the ⁇ -amylases from Bacillus licheniformis, from ⁇ . amyloliquefaciens or from ß. stearothermophilus and their further developments, especially for use in detergents and cleaning agents.
  • the ⁇ -amylase from Bacillus sp. A 7-7 DSM 12368
  • CGTase cyclodextrin glucanotransferase
  • amylolytic enzymes are in the focus of the present application, which belong to the sequence space of ⁇ -amylases, which is defined in the application WO 03/002711 A2, and those which are described in the application WO 03/054 * 177 A2. Fusion products of the molecules mentioned are also meant, for example those from the application DE 10138753 A1.
  • Lipases or cutinases can also be produced according to the invention, for example the lipases originally obtainable from Humicola lanuginosa (Thermomyces lanuginosus) or developed further, in particular those with the amino acid exchange D96L or the lipases or cutinases whose starting enzymes were originally isolated from Pseudomonas mendocina and Fusarium solanii.
  • cellulases that can be produced by natural producers are those from Bacillus sp. CBS 670.93 and CBS 669.93 as disclosed in WO 96/34092 A2.
  • further enzymes can be produced according to the invention, which are summarized under the term hemicellulases.
  • mannanases xanthan lyases
  • pectin lyases pectinases
  • pectin esterases pectate lyases
  • xyloglucanases xylanases
  • pullulanases ß-glucanases.
  • Detergent and cleaning agent enzymes also include oxidoreductases, for example oxidases, oxygenases, catalases, peroxides, such as halo-, chloro-, bromo-, lignin, glucose or manganese peroxidases, dioxygenases or laccases (Phenol oxidases, polyphenol oxidases) or all other enzymes described in the prior art for this area of application.
  • oxidoreductases for example oxidases, oxygenases, catalases, peroxides, such as halo-, chloro-, bromo-, lignin, glucose or manganese peroxidases, dioxygenases or laccases (Phenol oxidases, polyphenol oxidases) or all other enzymes described in the prior art for this area of application.
  • an important implementation of the invention is to use the PrfB factor to increase the yield in protein production by culturing bacteria by increasing the PrfB activity in the cells producing the protein. This happens, for example, by the fact that this factor is presented in the affected cells in a larger number or with a higher activity.
  • a preferred use is that in which the factor PrfB is derived from a factor of SEQ ID NOs 2, 4, 6, 8 or 10 and particularly preferably in the homology range of the PrfB of ⁇ mentioned at the outset. licheniformis falls or has the corresponding preferred properties.
  • Such use is advantageously achieved via the associated nucleic acids, for example by introducing them into the protein-producing cells or the precursor cells using molecular biological methods.
  • According to the invention is therefore a use of a nucleic acid coding for the factor PrfB to increase the yield in protein production by culturing bacteria by additionally expressing this gene.
  • nucleic acid coding for the factor PrfB being derived from a nucleic acid of SEQ ID NOs 1, 3, 5, 7 or 9; it particularly preferably falls into the described homology range of the prfB gene from ⁇ . licheniformis.
  • preferred uses according to the invention are additionally characterized in that they are based on a Transformation is based on the nucleic acid coding for the factor PrfB, preferably because of its stable establishment in the derived bacterial strains after the previous transformation.
  • preferred uses are characterized in that they are gram-positive bacteria, preferably of the genera Staphylococcus, Corynebacteria or Bacillus, in particular of the species Staphylococcus carnosus, Corynebacterium glutamicum, Bacillus subtilis, B. licheniformis, B. amyloliquefaciens , B. globigii or ß. lentus, and especially around strains of ß. licheniformis or ß. amyloliquefaciens.
  • uses according to the invention which are characterized in that they are gram-negative bacteria, preferably of the genera E. coli or Klebsiella, in particular strains of Escherichia coli K12, of Escherichia coli B or Klebsiella planticola, and very particularly of derivatives of Strains Escherichia coli BL21 (DE3), E. coli RV308, E. coli DH5 ⁇ , E. coli JM109, E coli XL-1 or Klebsiella planticola (Rf).
  • E. coli or Klebsiella preferably of the genera E. coli or Klebsiella, in particular strains of Escherichia coli K12, of Escherichia coli B or Klebsiella planticola, and very particularly of derivatives of Strains Escherichia coli BL21 (DE3), E. coli RV308, E. coli DH5 ⁇ , E. coli JM109, E coli XL-1
  • a performance-improved variant of a factor PrfB or a nucleic acid coding therefor is used, preferably one which is in one, particularly preferably both, positions in positions 71 and 100 of the mature protein according to SEQ ID NO. 2 correspond, which carries or codes for the amino acids asparagine (N) or serine (S).
  • the protein produced is a non-enzyme, preferably a pharmacologically relevant protein, particularly preferably for insulin or calcitonin.
  • the protein produced is an enzyme, preferably a hydrolytic enzyme or an oxidoreductase, particularly preferably one Protease, amylase, hemicellulase, cellulase, lipase, cutinase, oxidase, peroxidase or laccase.
  • an enzyme preferably a hydrolytic enzyme or an oxidoreductase, particularly preferably one Protease, amylase, hemicellulase, cellulase, lipase, cutinase, oxidase, peroxidase or laccase.
  • licheniformis which is available, for example, from the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1b, 38124 Braunschweig (http://www.dsmz.de) under order number 13, and to control chromosomal DNA from ß.
  • Mun ⁇ . licheniformis On the chromosomal DNA of ⁇ treated with the restriction enzyme Mun ⁇ . licheniformis, a single fragment of approximately 5.5 kB in size was identified, while the chromosomal DNA was digested by ⁇ . subtilis with Mun ⁇ die for ß. expected fragments.
  • the resistance encoded by the vector was selected.
  • the method of blue / white screening selection plates contained 80 ⁇ g / ml X-Gal was used to identify clones which had taken up a vector with an insert.
  • 200 clones were obtained, of which 5 clones could be identified by colony hybridization, which ß. Hcheniformis secA gene carried. These were checked by renewed Southern blot analysis with the probe described above and a vector derived from pHSG575 with which the secA gene of .beta. licheniformis bearing 5.5 kB Mun ⁇ fragment continued under the name pHMH1.
  • the cloned 5.5 kB area was initially characterized by restriction mapping.
  • single and double digests of pHMH1 were carried out with various enzymes and those fragments which carry parts of the sec / 4 / p.fß operon were identified by means of Southern blot analysis.
  • the resulting restriction map was supplemented after complete sequencing of the 5.5 kB fragment (see below) and can be seen in FIG. 4.
  • the 5.5 kB fragment shown in FIG. 4 was sequenced in partial sequences according to standard methods.
  • the partial sequences showed strong homologies to the following genes from ß. subtilis: fliT (coded for a flagella protein), orf189 / yvyD (unknown function), secA (translokase-binding subunit; ATPase) and prfB (peptide chain release factor 2), in exactly the same gene sequence as in ß. subtilis. These genes are also shown in Figure 4.
  • ß is derived from the application WO 91/02792 A1.
  • licheniformis strain from the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209, USA (http://www.atcc.org). There he is called ß. licheniformis ATCC 53926 and ß in connection with the present application.
  • licheniformis E It is originally from the ß strain via mutagenesis. licheniformis DSM 641 and is characterized by its advantageous culture and protein synthesis performance.
  • the p / f ⁇ gene from this ⁇ is analogous to the procedure in Example 1.
  • licheniformis E have been isolated and sequenced. It is under SEQ ID NO. 3 and in Figure 2 compared to the prfB from ß. licheniformis DSM 13 shown.
  • the prfB from this strain has some point mutations compared to the B. licheniformis DSM 13 strain, but in most cases these are silent mutations. Only the exchanges in positions 213 and 300 affect the amino acid sequence: At the amino acid level, this results in ß. licheniformis E opposite ß. licheniformis DSM 13 the two amino acid exchanges S71N and N100S. The relevant codons are highlighted in Figure 2 and the derived amino acids in Figure 1.
  • SEQ ID NO. 4 shows the relevant amino acid sequence. It is like that of FIG. 1, in turn, for the prfB of ⁇ . adapted licheniformis characteristic reading frame change in DNA position 73 (see. Example 1). Both SEQ ID NO. 4 and FIG. 1 thus show the complete amino acid sequence as it results after completely correct / n-v / Vo translation.
  • PrfB is actually an interesting candidate to influence protein synthesis in Bacillus licheniformis
  • the genes prfB from ß. licheniformis DSM13 and from ß. licheniformis E were each together with that for the secreted protease subtilisin from ß. lentus (BLAP), which has been described in the application WO 91/02792 A1, in the host strain ⁇ . licheniformis (DSM 13) expressed.
  • BLAP secreted protease subtilisin from ß. lentus
  • DSM 13 the extracellular to which subtilisin from ß serves as an indicator of protein production and secretion. protease activity due to lentus.
  • the prfB genes from ß were first used, starting from the respective chromosomal DNA. licheniformis DSM13 and from ß. licheniformis E amplified by PCR and cloned via the restriction sites Sa / I and BamH ⁇ in the Escherichia coli I Bacillus subtilis sbutie vector pCU3. Then a part of the chloramphenicol resistance cassette originally present in the vector pCU3 was cut out with the restriction enzymes Mun ⁇ and ⁇ / col and exchanged for an erythromycin resistance cassette. This erythromycin resistance cassette had previously been amplified by means of PCR and corresponding primers from the vector pE194.
  • pCU3Ery-p / fß The resulting construct pCU3Ery-p / fß is shown in Figure 6.
  • the respective gene prfB is under the control of a constitutive promoter. Indeed, depending on the prfB contained, there were two vectors, pCU3Ery-p / fß (DSM13) and pCU3Ery-p / fß (E).
  • the trunk ß. licheniformis DSM13 which had previously been transformed with the vector pCB56C mentioned in WO 91/02792 A1 (ß. licheniformis DSM 13 pCB56) and thus on an expression vector the subtilisin gene from ß. lentus (BLAP) was then transformed with these two constructs and for control with the pCU3Ery empty vector without p / fß.
  • subtilisin activities of the transformants obtained were cultivated in a shake flask at a constant pH of 7.2 over a period of 3 days. For this purpose, samples were taken after 48.5 and after 72.5 h to determine the subtilisin activities in the culture supernatant. The activity was determined photometrically using the peptide substrate Suc-Ala-Ala- Pro-Phe-pNA (AAPF). The result is summarized in the following Table 1: Table 1: Subtilisin activities of ß. licheniformis DSM13 pCB56C with simultaneous pfß expression.
  • the activity of the formed is determined in U / ml of the supernatant
  • Figure 1 Alignment of the PrfB from B. licheniformis E with the wild-type sequence of the PrfB from B. halodurans at the amino acid level.
  • PrfB_Baclich_ E Variant S71 N / N100S of the PrfB from ß. licheniformis
  • PrfB_Bachalo wild-type sequence of the PrfB from B. halodurans
  • PrfB have a homology of 63.2% identity at the amino acid level.
  • the wild type sequence of PrfB from ß. licheniformis has DSM 13 on both
  • Positions 71 and 100 represent the two amino acids S and N, respectively, resulting in the same homology values. These two positions are highlighted.
  • FIG. 2 Alignment of the DNA sequences of the PrfB from B. licheniformis E, that is to say the variant S71N / N100S according to the invention, with the wild-type sequence of the PrfB as obtained from B. licheniformis DSM 13.
  • prfB_Baclich_ E Sequence of the variant S71N / N100S from ß. licheniformis
  • EprfB_Baclich_DSM wild-type sequence of the PrfB from ß. licheniformis DSM 13
  • Consensus Consensus sequence The codons that relate to the two amino acid exchanges S71N and N100S are highlighted. The
  • Figure 3 Location of prfB in ß. licheniformis
  • the prfB gene is also in the sec / 4 region and a coherent mRNA is formed, so that one can also speak of a secA / fß operon.
  • Figure 4 Restriction map of the locus of prfB in B. licheniformis
  • Example 1 the gene prfB and an orf are located in the immediate vicinity of secA on an approximately 5.5 kB fragment which is obtained from the genomic DNA of ⁇ by restriction with Mun. licheniformis can be obtained.
  • Figure 5 Schematic representation of the translation / translocation apparatus of gram-positive bacteria.
  • PrfB acts on the detachment of the protein just formed from the ribosome.
  • Figure 6 The vector pCU3Ery-p / fß
  • this vector increases the protein formation rate via the constitutive expression of the gene prfB.

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Abstract

L'invention concerne le facteur PrfB (facteur de libération de chaîne peptidique B; RF2) issu de Bacillus licheniformis ainsi que l'ensemble des facteurs PrfB identiques à au moins 93 % sur le plan des acides aminés et au moins 80 % sur le plan de l'ADN. Ces facteurs PrfB, et en principe tous les facteurs PrfB, peuvent être employés pour augmenter le taux de croissance de protéines dans des procédés de fabrication de protéines au moyen de bactéries gram positives ou gram négatives, en particulier les facteurs PrfB dont les séquences sont données dans le descriptif. Ces facteurs PrfB sont issus des espèces B. licheniformis, B. halodurans, B. subtilis et Escherichia coli. L'invention concerne également l'utilisation du facteur PrfB dans l'augmentation du taux de croissance de protéines dans des procédés de fabrication de protéines au moyen de bactéries gram positives ou gram négatives. Les facterus PrfB dont l'activité est optimisée sont particulièrement adaptés. Un variant dont l'activité est amélioré est obtenu par échange des acides aminés S71N/N100S dans une position homologue à celle de la séquence du facteur PrfB sauvage issu de B. licheniformis.
PCT/EP2004/001950 2003-03-04 2004-02-27 Facteur prfb (facteur de liberation de chaine peptidique b) issu de bacillus licheniformis et utilisation de celui-ci pour l'augmentation du taux de formation de proteines WO2004085649A1 (fr)

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DE2003109555 DE10309555A1 (de) 2003-03-04 2003-03-04 Faktor PrfB (Peptide chain release factor B) aus Bacillus licheniformis und Steigerung der Proteinbildungsrate durch Erhöhung der PrfB-Aktivität
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012139964A1 (fr) * 2011-04-13 2012-10-18 Henkel Ag & Co. Kgaa Procédé d'expression

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HERBORT, M. ET AL.: "Temporal Expression of the Bacillus subtilis secA Gene, Encoding a Central Component of the Preprotein Translocase", JOURNAL OF BACTERIOLOGY, vol. 181, no. 2, January 1999 (1999-01-01), pages 493 - 500, XP002292890 *
KAROW, M.L. ET AL.: "Suppression of TGA Mutations in the Bacillus subtilis spoIIR Gene by prfB Mutations", JOURNAL OF BACTERIOLOGY, vol. 180, no. 16, August 1998 (1998-08-01), pages 4166 - 4170, XP002292889 *
UNO, M. ET AL.: "Functional specificity of amino acid at position 246 in the tRNA mimicry domain of bacterial release factor 2", BIOCHIMIE, vol. 78, no. 11-12, 1996, pages 935 - 943, XP002292888 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012139964A1 (fr) * 2011-04-13 2012-10-18 Henkel Ag & Co. Kgaa Procédé d'expression
CN103608457A (zh) * 2011-04-13 2014-02-26 巴斯夫欧洲公司 表达方法
US9663798B2 (en) 2011-04-13 2017-05-30 Basf Se Expression method

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