WO2004085649A1 - Prfb factor (peptide chain release factor b) from bacillus licheniformis and the utilization thereof for enhancing the protein formation rate - Google Patents

Abstract

The invention relates to factor PrfB (peptide chain release factor B; RF2) from Bacillus licheniformis and to all PrfB factors which are identical at the amino acid level by at least 93 % and at the DNA level by at least 80 %. These and in principle all PrfB can be used for enhancing the protein formation rate in methods for the production of proteins by means of gram-positive or gram-negative bacteria, more particularly those whose sequences have been prepared using the present application. This concerns those from the species B. licheniformis, B. halodurans, B. subtilis and Escherichia coli. The invention also relates to the additional preparation of factor PrfB for enhancing the protein formation rate in methods for the production of proteins by means of gram-positive or gram-negative bacteria. Particularly advantageous for said purpose are factors PrfB whose activity has been enhanced. A variant having an enhanced activity was found with amino acid exchanges S71N/N100S in an homologous position as that of the wildtype sequence of the PrfB from B. licheniformis.

Description

FACTOR PRFB (PEPTIDE CHAIN RELEASE FACTOR B) FROM BACILLUS LICHENIFORMIS AND THE USE THEREOF FOR INCREASING THE PROTEIN FOLLOWING RATE

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.

Genetic engineering processes for protein production have long been established in the prior art. For this purpose, the 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.

In the case of genetic engineering processes for protein production, the natural abilities of the microorganisms used for the production are first exploited for the synthesis and possibly for the secretion of the proteins. Basically, 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.

To increase the yield of protein formed which can be achieved from the respectively selected system, there are numerous complementary and predominantly also at the same time applicable strategies have been developed. These include, for example, optimizations that (1.) affect the physiological state of the host cells selected, such as the nutrient media composition or process parameters such as aeration (e.g. EP 211241 A2), (2.) genetic optimizations, such as adaptations in codon usage Increase in the number of copies of the transgene, for example by using so-called multicopynumber plasmids (for example DD 277468 A1), or increase in the lifespan of the associated mRNA and (3.) increase in the transcription and / or translation rate, for example by establishing suitable control elements. According to WO 92/19721 A2, for example, regulation is possible via factors or genes which act on the transcription.

This last-mentioned strategy therefore starts with the secretion of the proteins formed into the periplasm (in the case of gram-negative bacteria) or in the surrounding medium (both in the case of gram-negative and gram-positive bacteria), which is principally described in the article by A.J. Driessen (1994): "How proteins cross the bacterial cytoplasmic membrane" in J. Membr. Biol., Vol. 142 (2). Page 145-159. The secretion apparatus required for this consists of a number of different, mainly membrane-associated proteins. These include e.g. in Bacillus subtilis the proteins SecA, DF, E, G and Y (van Wely, KH, Swaving, J., Freudl, R., Driessen, AJ (2001): "Translocation of proteins across the cell envelope of Gram-positive bacteria ", FEMS Microbiol Rev. 2001, volume 25 (4), pages 437-454). A prerequisite for the translocation is that the proteins to be removed have a signal peptide at the N-terminal (Park, S., Liu, G., Topping, TB, Cover, WH, Randall, LL (1988): "Modulation of folding pathways of exported proteins by the leader sequence ", Science, volume 239, pages 1033-1035). A representation of the translocation apparatus supplemented with regard to the present application is shown in FIG. 5.

A recent study by B.v.d.Berg et al. (2004; Nature, volume 427, pages 36 to 44) describes the X-ray structure of the translokase, that is, the SecY complex from Methanococcus jannaschii as a model for the corresponding complex in other organisms.

The translocation is naturally preceded by protein synthesis, which is also indicated in FIG. 5. After the translation on the ribosomes, 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). 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. 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.

According to the teaching of WO 94/19471 A1, 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. These are systems that are not naturally associated with protein synthesis or translocation apparatus, but on the contrary indicate a rather pathological condition of the cell, for example the colicin or the Kil system.

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. (1999; "Temporal expression of the Bacillus subtilis secA gene, encoding a central component of the preprotein translocase", J. Bacteriol., Volume 1_81 (2), pages 493-500), the associated gene prfB in B. subtilis is behind the for the SecA factor second on a bicistronic operon. According to this article, both genes are coherent and maximally expressed towards the end of the exponential growth phase, which is associated with the highest secretion rate of exoenzymes. In the stationary growth phase, the education rate of SecA, and therefore also of PrfB, decreases significantly.

The work of Karow et al. (1998; "Suppression of TGA mutations in the Bacillus subtilis spollR gene by prfB mutations", J. Bacteriol., Volume 180 (16), pages 4166-70) discloses the amino acid sequences of the PrfB from the microorganisms B. subtilis, Streptomyces coelicolor, E. coli, Salmonella typhimurium and Haemophilus influenzae.

The publication "Sequence comparison of new prokaryotic and mitochondrial members of the polypeptide chain release factor family predicts a five-domain model for release factor structure" (HJPel et al. (1992); Nucl. Acids Res., Volume 20, pages 4423 bis 4428) refers to various peptide chain detachment factors B (PrfB) described in the prior art, in particular to the similarities between those which can be obtained from mitochondria and from prokaryotes, on the one hand regulating the expression of this gene in E. coli, Salmonella typhimurium and B. subtilis are represented by a reading frame mutation, and the sequences of the factors mRF-1 from mitochondria and RF-1 and RF-2 from B. 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. The 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/. For example, those from E. coli and Bacillus subtilis have the registration numbers Q8XD56 and NP_391409. However, the factor PrfB has not yet been described from B. licheniformis.

No commercial applicability has yet been proposed for PrfB, especially not for the molecules from B. licheniformis or highly homologous ones. In particular, 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. There is also no literature on the modification of PrfB by point mutagenesis and a possible increase in the synthesis rate of proteins produced by fermentation in this way.

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). Without wishing to be bound by this theory, one could assume that this effect is due to the fact that an increase in PrfB activity accelerates the detachment of newly formed amino acid chains from the ribosomes and the ribosomes in question are thus more quickly available for the synthesis of further proteins. Each individual mRNA should therefore be used to synthesize more proteins than compared to the normal intracellular PrfB activity level.

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.

As already explained in the introduction, factors PrfB from various microorganisms have already been described in the prior art. Their function in connection with protein biosynthesis is well known and is illustrated, for example, by FIG. 5. Surprisingly, the factor PrfB from Bacillus licheniformis has not yet been described.

Example 1 of the present application shows how, based on a known p / β sequence, that of an unknown PrfB can be found. In this case, 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. In particular, 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, as also explained in Example 1, is that there is a reading frame shift in the associated gene. For 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).

The DNA and amino acid sequences for 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.

The two factors subsequently disclosed in the sequence listing, but also generally accessible, as explained in the introduction, are the factors PrfB from E. coli and B. subtilis. They in turn have the reading frame mutation mentioned. In E coli the T in position 76 and in B. subtilis the T in position 73 are overlooked in order to synthesize a complete PrfB. This results in the SEQ ID NO. 8 or 10 specified amino acid sequences.

In all of these organisms, at least homology comparisons have shown that 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.

The searches in the publicly accessible databases (GenBank: National Center for Biotechnology Information NCBI, National Institutes of Health, Bethesda, MD, USA; EMBL-European Bioinformatics Institute (EBI) in Cambridge, Great Britain) with those according to Example 1 from ß. licheniformis DSM 13 sequences obtained the following on Next related genes or factors: The next similar gene is the prfB from ß. subtilis. Homology comparisons at the DNA and amino acid level revealed similarities of 78.5 and 92.4% identity, respectively. The second most similar factor described above is PrfB from ß. halodurans with 63% and the third most similar one from E. coli with 45.7% identity at the amino acid level.

For this reason, homologs with at least 93% identity at the amino acid level are claimed according to the invention. This includes both wild-type enzymes and variants that can be obtained, for example, through targeted or untargeted mutagenesis.

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. For this reason, within the homology range mentioned, 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.

Such factors are increasingly preferred 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.

Because they are all the more similar to the PrfB from β found and described in the examples as usable according to the invention. licheniformis DSM 13.

As a variant of 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.

As shown in Example 3, 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.

In example 1 the factor PrfB is not actually obtained from β. 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.

All factors PrfB that are encoded by a nucleic acid with a nucleotide sequence that corresponds to that in SEQ ID NO. 1 specified nucleotide sequence has a homology of at least 80% identity.

Increasingly preferred are those which are encoded by a 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.

According to what has been said above, this applies in particular to performance-improved variants of a factor PrfB, in particular those which are characterized in that the 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).

In accordance with these statements, 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.

Accordingly, 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.

Among these, the 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.

This applies in particular to 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).

In each case, very highly related genes according to the invention which are located in this protected area are obtained if a continuous reading frame is generated by deleting the nucleotide shifting the reading frame. This could, on the one hand, increase the rate of formation of the relevant factor and thus its intracellular activity and, on the other hand, predestine the gene in question for use in a microorganism which does not have such a reading frame shift within the prfB gene in vivo.

In preferred embodiments, 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.

This is because 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. In the case of expression vectors, it is additionally particularly advantageous if 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.

According to the above, 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.

Among these, those are preferred which are characterized in that they produce one of the factors PrfB described. On the one hand, this serves to obtain and biochemically characterize this protein. On the other hand, cells that produce a PrfB according to the invention and thus have a higher PrfB activity intracellularly than wild-type cells are particularly efficient with regard to the protein formation rate.

The last embodiment of 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.

As already mentioned above, 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.

This makes the PrfB factor economically interesting compared to its descriptions in the prior art, which have so far been of an academic nature. In addition to other possibilities that were described at the beginning, it provides another starting point by means of which the protein production can be improved in terms of its yield by cultivating bacteria. To date, improvements have been made which (1) affect the physiological state of the host cells selected, (2) genetic optimization, or (3) the increase in the rate of transcription, translation or translocation (see above). It is to be expected that the new starting point now made available can be combined with the previous techniques and thus yields which have been satisfactory to date can be further increased. In individual cases where one of the previous methods may not have been successful, this new approach represents a new alternative.

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.

Since the meaning of the factor PrfB has not been discussed previously for any of these methods, the implementation of the present invention is not restricted solely to the factors PrfB provided with the present application. In principle, 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.

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. In the case where the endogenous prfB is presented in a second identical copy (pCU3Ery-p / fß (DSM13)), 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.

Because the stable transformation with this performance-enhancing element makes it possible, as explained above, to produce production strains for a large number of different products of interest on the same principle. 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.

It is particularly advantageous to replace an endogenous PrfB, which is less active in the production strain that may be of interest for other reasons, with a more powerful PrfB. For example, 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. In addition, there is the possibility, for example, of introducing additional gene copies into the cell in question via the vectors set out above and thus achieving an additional increase in performance via the gene dose effect. 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.

Advantageously, 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.

Because of the great importance that gram-positive and certain gram-positive bacteria have in technical protein production, 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.

As stated above, 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.

Methods of this type which are particularly easy to implement because they are provided with the present application are characterized in that a 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.

This is because the relevant wild-type sequences are from the gram-positive microorganisms mentioned. In the case of another gram-positive bacterium, it still seems promising to try the increase in performance with one of the factors or genes specified here. Of these, performance-improved variants are advantageously used. In particular, the choice between the sequences from ß. halodurans and the other Bacilli offer a good starting point, because in the latter cases regulation is carried out in vivo via a reading frame shift, at ß. halodurans but not (see above). This peculiarity should advantageously correlate with the corresponding regulatory capabilities of the host organism. Otherwise, as already mentioned above, there is the possibility of generating a continuous reading frame via point mutagenesis and achieving corresponding advantages. Gram-negative bacteria are also used in particular for the characterization of genes and proteins on a laboratory scale, but also for large-scale production. Corresponding to their importance, corresponding protein production methods according to the invention are also embodiments of the present invention.

Such preferred methods with the properties described above are additionally characterized in that 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 DH5α, E. coli JM109, E. coli XL-1 or Klebsieila planticola (Rf).

According to what has been said for the gram-positive organisms, 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.

Accordingly, 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 / fβ sequence from the gram-negative model organism E. coli. In accordance with what has been said for the other sequences, 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.

As already mentioned, performance-improved variants of PrfB are of particular interest according to the invention. As such a variant, 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).

A large number of technical protein production processes are based on the factors naturally formed by the microorganisms in question. For example, the economically important subtilisins have been found as such factors, those of Bacilli that start with ß. subtilis are related, formed and secreted. So if not only the genes in question are present but also read, these are preferred methods.

Accordingly, correspondingly preferred methods are characterized in that 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.

In contrast, protein production processes based on so-called transgenes, that is to say those which have been introduced into suitable host cells and are expressed by them, are becoming increasingly important. 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.

Accordingly preferred methods are characterized in that 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.

It is particularly advantageous both for endogenously coded proteins and for those which are derived from transgenes if they can be isolated from the surrounding culture medium and thus purified. For example, the increase was also possible in Example 3 the protein formation rate can be determined by measuring the activity in question in the supernatant. However, 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.

Accordingly, preferred methods are characterized in that the protein produced is secreted.

Of particular interest are methods according to the invention which are aimed at certain products produced by the cultivation of the microorganisms. Accordingly, preference is given to 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.

But 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. Thus, according to the invention, 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. Of these, the representatives that are preferably produced for use in detergents and cleaning agents are listed below.

In principle, all such enzymes established in the prior art can be used to increase the washing or cleaning performance. Among the 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. Further proteases from various Bacillus sp. and ß. gibsonii go from the patent applications WO 03/054185 A1, WO 03/056017 A2, WO 03/055974 A2 and. WO 03/054184 A1. Examples of amylases which can be produced according to the invention are the α-amylases from Bacillus licheniformis, from β. amyloliquefaciens or from ß. stearothermophilus and their further developments, especially for use in detergents and cleaning agents. Furthermore, for this purpose, the α-amylase from Bacillus sp. A 7-7 (DSM 12368) and the cyclodextrin glucanotransferase (CGTase) described in the application WO 02/44350 A2 from β. to highlight agaradherens (DSM 9948). Furthermore, the amylolytic enzymes are in the focus of the present application, which belong to the sequence space of α-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.

It is also intended in particular for the technical treatment of textiles, but also for detergents on cellulases, for example endoglucanase and / or cellobiohydrolases. For example, 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. Furthermore, further enzymes can be produced according to the invention, which are summarized under the term hemicellulases. These include, for example, mannanases, xanthan lyases, pectin lyases (= pectinases), pectin esterases, pectate lyases, xyloglucanases (= xylanases), pullulanases and 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.

The present invention is realized not only by the substances and methods described so far, but also by certain possible uses of the substances mentioned. They are also included in the scope of the present application in accordance with the previous statements.

Thus, 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 β 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.

It is advantageous to use the sequences described in the present application and recognized as correspondingly favorable. Accordingly, any such use of a nucleic acid is preferred, the 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 β. licheniformis.

According to the molecular biological possibilities, 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.

Because of the importance of gram-positive bacteria shown above, 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.

The same applies to 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 DH5α, E. coli JM109, E coli XL-1 or Klebsiella planticola (Rf).

Further preferred, analogous to what has been said above, are such uses, which are characterized in that 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).

Since such uses are particularly relevant in connection with the production of economically important proteins, those uses are particularly preferred which are characterized in that the protein produced is a non-enzyme, preferably a pharmacologically relevant protein, particularly preferably for insulin or calcitonin.

The same applies to such uses, which are characterized in that 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.

Examples

All molecular biological work steps follow standard methods, such as those given in the manual by Fritsch, Sambrook and Maniatis "Molecular cloning: a laboratory manual", Cold Spring Harbor Laboratory Press, New York, 1989, or comparable relevant works. Enzymes and building kits (kits) were used according to the information of the respective manufacturers.

example 1

Isolation of the gene prfB from ß. licheniformis

Identification of the prfiB locus in B. licheniformis

To identify the sec> 4 / p / 7B locus in ß. licheniformis was a gene probe via PCR based on the known sequence of the p / fß-sec.4 gene location of ß. subtilis (database "Subtilist" of the Institut Pasteur, 25.28 rue du Docteur Roux, 75724 Paris CEDEX 15, France; http://genolist.pasteur.fr/SubtiList; as of August 16, 2002). This locus is also shown in Figure 3. The probe obtained was 3113 bp long and additionally comprised the first 451 bp of the N-terminal region of the prfB gene. Then preparations of chromosomal DNA from ß. 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. Subtilis digested with various restriction enzymes and subjected to Southern hybridization with the probe mentioned. On the chromosomal DNA of β 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 β. subtilis with Mun \ die for ß. expected fragments.

Cloning of the identified area from B. licheniformis DSM13

Chromosomal DNA of the same strain ß. licheniformis was isolated, preparatively digested with Mun \ and the DNA region around 5.5 kB isolated using agarose gel electrophoresis and the nucleic acids extracted therefrom using commercially available kits. The resulting mixture of Mu / il-cut DNA fragments was described in the Mun \ compatible EcoRI site of the low copy number vector pHSG575 (described in: "High-copy-number and low-copy-number vectors for lacZ alpha-complementation and chloramphenicol- or kanamycin-resistance selection"; S. Takeshita; M. Sato; M. Toba; W. Masahashi; T. Hashimoto-Gotoh; Gene (1987), volume 61, pages 63-74) and transformed into E. coli JM109 (available from Promega, Mannheim, Germany).

The resistance encoded by the vector was selected. In addition, the method of blue / white screening (selection plates contained 80 μg / ml X-Gal) was used to identify clones which had taken up a vector with an insert. In addition, 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.

restriction analysis

The cloned 5.5 kB area was initially characterized by restriction mapping. For this purpose, 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.

sequence analysis

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.

An open reading frame in the range of Bp 4177-5278 was also found in the sequence which codes for a 366 amino acid protein. In the area -14 to -8 at the start of translation there is homologous to the corresponding area in the ß. subtilis genome a possible ribosome binding site (GAGGTGA). This from ß. licheniformis DSM 13 determined DNA sequence is under SEQ ID NO. 1 shown; the deduced amino acid sequence comes from SEQ ID NO. 2 out.

It should be noted that in numerous species, and obviously also in ß. licheniformis, the expression of prfB is regulated in a special way: the reading frame is changed within the gene. For this reason, the number in SEQ ID NO. 1 specified positions by 1 higher than a multiple of three. When this factor is completely // 7 Vo synthesized, the first 72 nucleotides, i.e. those which code for the first 24 amino acids, are correctly transcribed, then the T is skipped at position 73 and the transcription from position 74 is included to generate a continuous reading frame the codon GAC (corresponding to Asp 25) continued until the end of the gene. In SEQ ID NO. 2 shows the connected amino acid sequence of the PrfB from B. licheniformis DSM 13, as was obtained according to this example. This sequence and in particular the change in the reading frame are supported by homology comparisons with the sequences of other factors PrfB from the relevant databases.

Research in publicly available databases (GenBank: National Center for Biotechnology Information NCBI, National Institutes of Health, Bethesda, MD, USA; EMBL-European Bioinformatics Institute (EBI) in Cambridge, Great Britain) revealed the prfB from ß as the next similar gene. subtilis. Homology comparisons at the DNA and amino acid level revealed similarities of 78.5 and 92.4% identity, respectively. For the PrfB from ß. Halodurans were determined at the amino acid level for 63% and for 45.7% identity from E. coli.

Based on these values, it can also be assumed that the PrfB from ß. licheniformis the same biochemical activity as in particular the PrfB of ß. exercises subtilis and thus assumes the same physiological function.

Example 2

The variant S71N / N100S

A special 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 / fβ gene from this β 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. As can be seen in FIG. 2, 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.

Also SEQ ID NO. 4 shows the relevant amino acid sequence. It is like that of FIG. 1, in turn, for the prfB of β. 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.

It stands to reason that the good protein synthesis performance of the strain ß. licheniformis E can be attributed at least in part to these two amino acid exchanges S71N and N100S in factor PrfB.

Example 3

Measurement of the rate of formation of extracellular protease with simultaneous

Expression of the p / fß gene

In order to investigate whether 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 β. licheniformis (DSM 13) expressed. In this system, the extracellular to which subtilisin from ß serves as an indicator of protein production and secretion. protease activity due to lentus.

For this purpose, 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 Λ / 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. 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.

To determine the prfB effect, the 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

Subtilisin.

These values show that the additional expression of the factor PrfB from the wild-type strain DSM 13 by the plasmid pCU3Ery-p / fß (DSM13), that is to say by the intracellular increase in the PrfB activity, already increases the product formation rate. An additional increase can be seen when using the factor PrfB S71N / N100S as it is formed from the plasmid pCU3Ery-pr B (E).

Description of the figures

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.

Where:

PrfB_Baclich_ E: Variant S71 N / N100S of the PrfB from ß. licheniformis

PrfB_Bachalo: wild-type sequence of the PrfB from B. halodurans

Consensus: consensus sequence

These two 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.

Therein: 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

Differences can be traced back to a changed nucleotide in positions 213 and 300.

Figure 3: Location of prfB in ß. licheniformis

It can be seen that 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

As shown in 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 β by restriction with Mun. licheniformis can be obtained. Figure 5: Schematic representation of the translation / translocation apparatus of gram-positive bacteria.

According to van Wely, K.H., Swaving, J., FreudI, R., Driessen, A.J. (2001); "Translocation of proteins across the cell envelope of Gram-positive bacteria", FEMS Microbiol Rev. 2001, Volume 25 (4), pages 437-54; PrfB has been added. As shown by an arrow, PrfB acts on the detachment of the protein just formed from the ribosome.

Figure 6: The vector pCU3Ery-p / fß

As described in Example 3, this vector increases the protein formation rate via the constitutive expression of the gene prfB.

Claims

claims 1. 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. 2. Factor PrfB according to claim 1 with an amino acid sequence which corresponds to that in SEQ ID NO. 2 amino acid sequence increasingly preferred has a homology of 94%, 95%, 96%, 97%, 98%, 99% identity and particularly preferably 100% identity. 3. Variant of a factor PrfB according to claim 1 or 2, characterized in that in one, preferably both positions, the positions 71 and 100 of the mature protein according to SEQ ID NO. 2 correspond, the amino acids asparagine (N) or serine (S) are present. 4. Factor PrfB, encoded by a nucleic acid with a nucleotide sequence that corresponds to that in SEQ ID NO. 1 specified nucleotide sequence has a homology of at least 80% identity. 5. Factor PrfB according to claim 4, encoded by a nucleic acid with a nucleotide sequence which corresponds to that in SEQ ID NO. 1 indicated nucleotide sequence increasingly preferably has a homology of 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5% identity and particularly preferably 100% identity. 6. Variant of a factor PrfB according to claim 4 or 5, characterized in that the nucleic acid in question for one, preferably both positions, the positions 71 and 100 of the mature protein according to SEQ ID NO in a homologous position. 2 correspond, encoded for the amino acids asparagine (N) or serine (S). 7. Nucleic acid coding for a factor PrfB with a nucleotide sequence which corresponds to that in SEQ ID NO. 1 specified nucleotide sequence has a homology of at least 80% identity. 8. A nucleic acid coding for a factor PrfB according to claim 7 with a nucleotide sequence which corresponds to that in SEQ ID NO. 1 indicated nucleotide sequence increasingly preferably has a homology of 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5% and particularly preferably 100% identity. 9. A nucleic acid coding for a factor PrfB according to claim 7 or 8, characterized in that it for one, preferably both positions, in positions 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). 10. Vector which contains a nucleic acid according to one of claims 7 to 9, in particular one which codes for a factor PrfB according to one of claims 1 to 6. 11. Vector according to claim 10, characterized in that it is a cloning vector and / or an expression vector, preferably one that can be kept stable in the derived bacterial strains after transformation. 12. A cell that has been obtained by transformation with a nucleic acid according to one of claims 7 to 9 or is derived from such a cell, in particular by transformation with a vector according to one of claims 10 or 11. 13. Cell according to claim 12, characterized in that it produces a factor PrfB according to one of claims 1 to 6. 14. A method for producing a factor PrfB according to one of claims 1 to 6 using a nucleic acid according to one of claims 7 to 9, particularly using a vector according to one of claims 10 or 11, very particularly preferably using a cell according to one of the Claims 12 or 13. 15. A method for protein production by culturing bacteria, characterized in that the factor PrfB (peptide chain release factor B) is present in the cells producing the protein in a higher activity than naturally in these cells. 16. The method according to claim 15, characterized in that the higher PrfB activity is due to additional expression of the gene coding for this factor PrfB prfB. 17. The method according to claim 16, characterized in that the additional expression of the factor PrfB is due to additional copies of the gene pyfß in the genome of the bacteria concerned, preferably due to their stable establishment in the derived bacterial strains after previous transformation. 18. The method according to any one of claims 15 to 17, characterized in that in the protein-producing cells, the endogenous factor PrfB has been replaced by a PrfB with higher activity, preferably with replacement of at least one endogenously present p / fß gene by the for p / fβ gene encoding the PrfB with higher activity. 19. The method according to any one of claims 15 to 18, characterized in that the additional PrfB activity is achieved by a factor PrfB according to one of claims 1 to 6, preferably with the aid of a nucleic acid according to one of claims 7 to 9, particularly preferably with Help of a vector according to one of claims 10 or 11, very particularly preferably by a cell according to one of claims 12 or 13. 20. The method according to any one of claims 15 to 19, characterized in that the cells producing the protein are gram-positive bacteria, preferably of the genera Staphylococcus, Corynebacteria or Bacillus, in particular the species Staphylococcus carnosus, Corynebacterium glutamicum, Bacillus subtilis, B licheniformis, B. amyloliquefaciens, B. globigii or B. lentus, and especially around strains of ß. licheniformis or ß. amyloliquefaciens. 21. The method according to claim 20, 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. 22. The method according to claim 20 or 21, 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. 23. The method according to any one of claims 20 to 22, characterized in that a PrfB is used which is based on the wild-type sequence from ß. licheniformis, B. halodurans or ß. subtilis, preferably from 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. 24. The method according to any one of claims 15 to 19, characterized in that the cells producing the protein 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 especially derivatives of the strains Escherichia coli BL21 (DE3), E coli RV308, FS. coli DH5α, E. coli JM109, E. coli XL-1 or Klebsiella planticola (Rf). 25. The method according to claim 24, 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. 26. The method according to claim 24 or 25, 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. 27. The method according to any one of claims 24 to 26, 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. 28. The method according to any one of claims 15 to 27, characterized in that an activity-improved variant of a factor PrfB is used, preferably one which is in one, particularly preferably two positions, which is in homologous positions 71 and 100 of the mature protein according to SEQ ID NO. 2 correspond to the amino acids asparagine (N) or serine (S). 29. The method according to any one of claims 15 to 28, characterized in that 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. 30. The method according to any one of claims 15 to 29, characterized in that the gene coding for the protein produced has been introduced by transformation into the progenitor cells of the bacteria used for the production, preferably via an expression vector, particularly preferably because of its stable establishment in the derived bacterial strains. 31. The method according to claim 29 or 30, characterized in that the protein produced is secreted. 32. The method according to any one of claims 15 to 31, characterized in that the protein produced is a non-enzyme, preferably a pharmacologically relevant protein, particularly preferably insulin or calcitonin. 33. The method according to any one of claims 15 to 31, 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. 34. Use of the PrfB factor to increase the yield in protein production by culturing bacteria by increasing the PrfB activity in the cells producing the protein. 35. Use according to claim 34, wherein the factor PrfB is derived from a factor of SEQ ID NOs 2, 4, 6, 8 or 10, particularly preferably according to one of claims 1 to 6. 36. 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. 37. Use according to claim 36, wherein the nucleic acid coding for the factor PrfB is derived from a nucleic acid of SEQ ID NOs 1, 3, 5, 7 or 9, particularly preferably according to one of claims 7 to 9. 38. Use according to one of claims 34 to 37, characterized in that it is based on a transformation with the nucleic acid coding for the factor PrfB, preferably because of its stable establishment in the derived bacterial strains after the previous transformation. 39. Use according to one of claims 34 to 38, characterized in that it is gram-positive bacteria, preferably of the genera Staphylococcus, Corynebacteria or Bacillus, in particular 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. 40. Use according to one of claims 34 to 38, characterized in that it is Gram-negative bacteria, preferably of the genera E. coli or Klebsiella, in particular strains of Escherichia coli K12, Escherichia coli B or Klebsiella planticola, and very particularly derivatives of the strains Escherichia coli BL21 (DE3), E. coli RV308, E. coli DH5α, E. coli JM109, E. coli XL-1 or Klebsiella planticola (Rf). 41. Use according to one of claims 34 to 40, characterized in that 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 in a homologous position of the mature protein according to SEQ ID NO. 2 correspond, which carries or codes for the amino acids asparagine (N) or serine (S). 42. Use according to one of claims 34 to 41, characterized in that the protein produced is a non-enzyme, preferably a pharmacologically relevant protein, particularly preferably insulin or calcitonin. 43. Use according to one of claims 34 to 42, 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.