Secretory production of recombinant proteins using bacterial fnr mutants of the family enterobacteriaceae

Abstract

The invention relates to a method for the fermentative production of recombinant proteins using fnr mutants of E. coli. Using these strains, increased amounts of recombinant protein in the culture medium can be achieved compared to the prior art.

Description

[0001] Co 12404 / Reu

[0002] Secretory production of recombinant proteins using bacterial fnr mutants of the Enterobacteriaceae family

[0003] The invention relates to a process for the secretory production of recombinant proteins using fnr mutants of Escherichia coli, characterized in that a bacterial strain from the family Enterobacteriaceae with attenuated Fnr activity containing at least one gene encoding a recombinant protein, which is functionally linked to a type II signal sequence encoding a signal peptide for transport into the periplasm, is used, the recombinant protein is produced by these bacteria in a fermentative process and secreted into the medium, and the recombinant protein is isolated.

[0004] The market for recombinant protein pharmaceuticals (pharmaceutical proteins / biologics) has grown significantly in recent years. Particularly important protein pharmaceuticals are eukaryotic proteins, especially mammalian and human proteins. Examples of important pharmaceutical proteins (pharmaceutically active proteins) include cytokines, growth factors, protein kinase, protein and peptide hormones, as well as antibodies and antibody fragments. Due to the still very high production costs for pharmaceutical proteins, there is a continuous search for more efficient and therefore more cost-effective methods and systems for their manufacture.

[0005] Generally, recombinant proteins are produced either in mammalian cell cultures or in microbial systems. Microbial systems have the advantage over mammalian cell cultures that recombinant proteins can be produced in a shorter time and at a lower cost. Bacteria are therefore particularly suitable for the production of recombinant proteins. Due to its very well-studied genetics and Co 12404 / Reu

[0006] 2

[0007] Due to its physiology, short generation time, and ease of handling, the Gram-negative enterobacterium Escherichia coli (E. coli) is currently one of the most frequently used organisms for the production of recombinant proteins. Production methods in which the target protein is released directly into the fermentation medium from the bacterial cells in its correct folded state are particularly attractive, as this eliminates the need for complex cell disruption and potentially lossy protein refolding. A further advantage of extracellular production is that releasing the target protein into the culture medium often increases the product yield, since the accumulation of the target protein is not limited to the periplasm or cytoplasm.

[0008] E. coli belongs to the Enterobacteriaceae family and, like all members of this family, is a facultatively anaerobic microorganism, meaning it can grow in both aerobic and anaerobic environments. For this to occur, the cells must be able to adapt to changes in growth conditions. In E. coli, the DNA-binding protein Fnr (fumarate nitrate reduction regulatory protein) plays a significant role in regulating and coordinating metabolism during the transition from aerobic to anaerobic growth conditions. As a transcriptional regulator, it controls the expression of hundreds of genes. Generally, this protein activates genes required for anaerobic metabolism and suppresses genes involved in aerobic metabolism. Furthermore, Fnr also regulates the transcription of many genes with other functions, such as acid resistance, chemotaxis, cell structure, and molecular biosynthesis.

[0009] The DNA sequence shown in SEQ ID No. 1 comprises the fnr wild-type gene of Escherichia coli K12, with the sequence of nucleotide 150-902 encoding the Fnr protein with the sequence SEQ ID No. 2. Synonymous names for the fnr gene are frdB, nirA, nirR, ossA, or oxrA. The cellular concentration Co 12404 / Reu

[0010] The activity of Fnr is similar in anaerobic and aerobic growth, but it is directly regulated by oxygen. Under anaerobic conditions, a [4Fe-4S] cluster is incorporated into Fnr, causing a conformational change and dimerization of the protein, thereby activating it. The presence of oxygen leads to the inactivation of Fnr through oxidation of this [4Fe-4S] cluster to a [2Fe-2S] cluster and dissociation of the dimer. Under anaerobic conditions, the expression of the fnr gene is subject to negative autoregulation by the active [4Fe-4S] cluster-containing Fnr protein.

[0011] The fnr gene is not absolutely essential for cells, and thus a number of different mutations in the Anr gene locus of E. coli are known to lead to a complete or partial loss of Fnr activity. This results, among other things, in the cells no longer being able, or only being able to grow to a limited extent, under anaerobic conditions using fumarate as an electron acceptor. In fact, the naming of the fnr gene locus is based on the observation that certain mutants of this genome region exhibit a defect in fumarate and nitrate reduction.

[0012] US 2008 / 057034 AA describes enterobacteria that exhibit an attenuating mutation in the fnr gene, whereby an attenuating mutation can result in either a reduction or complete loss of the activity of the Fnr regulatory protein. In the pathogenic strain Salmonella enteri ca serovar Typhlmurl um, a transposon insertion between base pairs 106 and 107 leads to the inactivation of the fnr gene, which is phenotypically expressed, among other things, in restricted cell motility due to the absence of flagella, as well as in an avirulent phenotype. Such Fnr-deficient and therefore attenuated enterobacteria with respect to their pathogenicity can, for example, be used as an attenuated live vaccine to elicit an immune response against these or other closely related species. Genera of Enterobacteriaceae from soluble . Co 12404 / Reu

[0013] 4

[0014] According to US 2008 / 057034 AA, these attenuated cells can also serve as vectors that produce an antigen heterologous with respect to the enterobacterium (e.g., a specific surface protein of a pathogenic organism) and, when introduced into another organism, trigger an immune response against this antigen.

[0015] Instead of producing an antigen to stimulate an immune response, Fnr-deficient strains can also be used to deliver therapeutic proteins into a host. For this purpose, the nucleic acids encoding the antigen or therapeutic protein, along with the corresponding elements necessary for expression (e.g., promoter, etc.), are either integrated into the bacterial cell's chromosome or contained on a plasmid. The heterologous protein is synthesized either as part of a fusion with a structural protein, such as a flagellin or a protein of the bacterial host's outer membrane, and is then presented anchored to the bacterial cell surface.Alternatively, the foreign antigen can also be linked to a suitable signal sequence that enables the secretion of the fusion protein from the cytoplasm of the bacterial cell directly into the extracellular space, where the target protein is intended to exert its antigenic effect.

[0016] According to US 2008 / 057034 AA, the key characteristic of f nr-attenuated cells is their reduced virulence and the resulting suitability for use as a live vaccine.

[0017] It is known that Escherichia coli is naturally unable to secrete proteins in large quantities directly into the extracellular space (Kleiner-Grote et al., 2018, Eng Li fe Sec. 18, pp. 532-50). Therefore, it is necessary either to produce the target protein as a fusion protein including a specific signal sequence or to make targeted modifications to the cell envelope of the production strain used, with the technical disadvantages described below.

[0018] For the one-step secretion of a recombinant protein from the cytoplasm directly into the extracellular space, i.e., without Co 12404 / Reu

[0019] 5. Since the protein is temporarily present in free form in the periplasm, signal sequences from type I or type ITT secretion systems are particularly suitable for Enterobacteriaceae (Kleiner-Grote et al., so). For example, a fusion protein consisting of a truncated version of the circumsporozoite protein of Plasmodium falciparum (tCSP) and the C-terminal domain of the cytolysin protein ClyA (HlyA, SheA) of Salmonella serovar Typhi, which serves as a type I signal sequence, can be transported directly into the extracellular space.

[0020] However, using a signal sequence from a type I secretion system for the secretory production of a recombinant target protein with an enterobacterium has the significant disadvantage that the signal sequence remains bound to the target protein after secretion. Therefore, obtaining the pure target protein requires targeted cleavage of the signal sequence, thus necessitating an additional processing step.

[0021] Another way to release recombinant proteins from E. coli is to use so-called "leaky" strains, which, due to the absence or alteration of certain structural elements of the cell membrane, release increased amounts of proteins located in the periplasm into the medium. Such "leaky" strains may exhibit altered lipoprotein components in the outer membrane, as is the case, for example, with certain mutants of Braun's lipoprotein (Ipp) (Suzuki 1978, Mol. Gen. Genet. 167, pp. 1-9).

[0022] To enable the release of the recombinant protein across the defective membrane, the protein must first be transported from the cytoplasm into the periplasm (Kleiner-Grote et al., so). For this type of two-step secretion process, the target protein must be functionally linked to a signal peptide that is recognized by certain proteins (e.g., SecB or SRP) of the type II secretion system and then directed by them to the Sec translocase. The Sec translocase is a multiprotein complex integrated into the cytoplasmic membrane, Co 12404 / Reu

[0023] 6 through which the transport of the fusion protein into the periplasm takes place. Even during the translocation of the protein across the cell membrane, the signal peptide is cleaved from the fusion protein by a cell-specific protease, which represents a major advantage over a process using a type I secretion system.

[0024] These are production processes for recombinant proteins on an industrial scale, in which, with the help of ipp mutants of E. coli, a release of target proteins into the fermentation medium is achieved (US 2008 / 0254511 Al ).

[0025] Due to their higher propensity for cell lysis, "leaky" strains have the disadvantage in the production of some heterologous target proteins that, despite certain measures to stabilize the cells, such as the addition of increased amounts of Ca and Mg ions to the culture medium (see US 2008 / 0254511 Al), they lyse relatively early and extensively. This shortens the protein production phase and thus results in a lower product yield than would be possible with a longer production phase. Furthermore, cell lysis leads to an increase in the viscosity of the medium, primarily due to the DNA released during cell lysis. This complicates the subsequent purification and recovery of the target protein, leading to unnecessarily high processing costs.

[0026] The object of the invention is to provide a fermentative process for the production of a recombinant protein using a bacterial strain, in which the recombinant protein is effectively excreted into the fermentation medium.

[0027] This problem is solved by a process for the fermentative production of recombinant proteins characterized in that a bacterial strain from the family Enterobacteriaceae with attenuated Fnr activity containing at least one gene encoding a recombinant protein which is functionally linked to a type I I signal sequence encoding Co 12404 / Reu

[0028] 7 a signal peptide for transport into the periplasm, the recombinant protein is produced by these bacteria in a fermentative process and secreted into the medium and the recombinant protein is isolated .

[0029] Surprisingly, it was found in the present invention that a bacterial strain of the family Enterobacteriaceae with attenuated Fnr activity secretes a recombinant target protein, equipped with a signal peptide suitable for a type II secretion system, into the culture medium in a higher quantity than an otherwise genetically identical fnr wild-type strain and in some cases even more than known “leaky” strains such as Ipp mutants.

[0030] A particular advantage of bacterial strains with attenuated Fnr activity in the production of a recombinant target protein is that the amount of target protein present in the culture medium is higher than in fnr wild-type strains.

[0031] This is clearly demonstrated by Example 2 (see Table 1). The anti-CD154 Fab titer measured in the culture supernatant was, in absolute terms (i.e., normalized to the same volume), at least more than twice as high when using Afnr mutants as when using the corresponding fnr wild-type strains.

[0032] Example 3 (Table 2) also confirms that when using Afnr mutants, significantly higher CGTase levels were measured in the culture supernatant than in the corresponding fnr wild-type strains.

[0033] Another advantage of bacterial strains with attenuated Fnr activity in the production of a recombinant target protein is that the majority of the target protein is excreted into the fermentation medium during cultivation. This is clearly demonstrated in Example 2 (see Table 1). While at least 38% of the anti-CD154 Fab titer was measured in the culture supernatant when using the Afnr mutants, it was only just under 33% when using the known Ipp3 mutant (JE5512 lpp3 / pJF118ut-CD154) and the wild-type strain Co 12404 / Reu.

[0034] 8

[0035] JE5512 / pJF118ut-CD154 only about 6%. In Example 3, using an lpp3 Afnr mutant, approximately 75% of the recombinant target protein (CGTase) could be measured in the culture supernatant.

[0036] Within the scope of the present invention, attenuated Fnr activity of a bacterial strain means that the strain has a reduced Fnr activity compared to the same strain before modification of its Fnr activity.

[0037] Within the scope of this invention, protein names such as Fnr begin with a capital letter, while the sequences encoding these proteins (cds) begin with a lowercase letter and are written in italics (e.g. fnr).

[0038] Methods for determining the Fnr activity of a strain are known to those skilled in the art (Williams et al., 1991, Nucleic Acids Res 19, pp. 6705-12; Williams et al., 1997, Nucleic Acids Res 25, pp. 4028-34). For example, the determination of β-galactosidase activity serves as a measure of the Fnr activity of a cell, where the β-galactosidase gene is functionally linked to the promoter sequence of a gene (e.g., ndh) whose expression is naturally controlled by the transcriptional regulator Fnr.

[0039] Approaches to attenuate Fnr activity in a strain of microorganisms are known in the prior art.

[0040] In a preferred embodiment, the method is characterized in that the attenuated Fnr activity is caused by the presence of at least one mutation in the coding region of the Fnr gene. That is, the Fnr activity is reduced by the presence of at least one substitution, insertion, or deletion of one or more nucleotides in the ORF of the Fnr gene.

[0041] In an alternative preferred embodiment, the method is characterized in that the attenuated Fnr activity is caused by a reduction in the expression of the Fnr gene. That is, the Fnr activity is reduced because Co 12404 / Reu

[0042] 9. A smaller amount of the Fnr protein is produced by the cell. This is particularly favored when at least one element necessary for expression regulation (e.g., promoter, enhancer, ribosome binding site) contains at least one mutation (substitution, insertion, or deletion of one or more nucleotides). Such DNA sequences, which differ in their base sequence from that of the Anr wild-type gene due to mutations, are referred to below as .fnr alleles.

[0043] The ORF of the fnr gene is particularly preferred, especially when the entire fnr gene is completely deleted.

[0044] A preferred method for the fermentative production of recombinant proteins is characterized in that the attenuated Fnr activity is caused by the fact that the fnr gene is an allele of SEQ ID No. 1, wherein the sequence of the .fnr allele is at least 80%, particularly preferably at least 90% and especially preferably at least 95% identical to the sequence of SEQ ID No. 1.

[0045] Methods for generating Anr alleles are known to those skilled in the art. Alleles of the fnr gene can be produced, for example, by nonspecific or targeted mutagenesis using the DNA of the Anr wild-type gene as starting material. Nonspecific mutations within the fnr gene or the promoter region of the fnr gene can be generated, for example, by chemical agents such as nitrosoguanidine, ethyl methanesulfonic acid, etc., and / or by physical methods and / or by PCR reactions carried out under specific conditions. Methods for introducing mutations at specific positions within a DNA fragment are known. For example, the exchange of one or more bases in a DNA fragment comprising the fnr gene and its promoter region can be achieved by PCR using suitable oligonucleotides as primers. Furthermore, it is possible to modify the entire fnr gene or... to produce a new fnr allele by gene synthesis. Co 12404 / Reu

[0046] Ten fnr alleles are typically first generated in vitro and then inserted into the cell's chromosome, thereby replacing the originally present Anr wild-type gene and creating an Anr mutant of the strain. fnr alleles can be introduced into a cell's chromosome in place of the Anr wild-type gene / promoter using known standard methods. This can be achieved, for example, using the method described in Link et al. (1997, J. Bacteriol. 179, pp. 6228-37) for introducing chromosomal mutations into a gene via the mechanism of homologous recombination. The introduction of a chromosomal deletion of the entire fnr gene or part thereof is possible, for example, using the X-Red recombinase system according to the method described by Datsenko and Wanner (2000, Proc. Natl. Acad. Sci. US A. 97, p. 6640-5). fnr alleles can also be introduced via transduction using Pl phages or conjugation from a strain with an Anr mutation onto a [missing information].The fnr wild-type strain is transferred, whereby the Anr wild-type gene in the chromosome is replaced by the corresponding Anr allele. In addition to the described E. coli strains with an Anr mutation, methods for generating Anr mutants of any E. coli strain are also known to those skilled in the art.

[0047] A preferred Anr mutation is a mutation that leads to at least partially attenuated Fnr activity. Examples include mutations that result in at least one of the following amino acid substitutions in the Fnr protein: D43G, R72H, S73F, I81T, T82P, G85A, D86A, E87K, Q88E, T118A, T118P, M120I, M120R, M120T, M120V, M144A, M147A, I151A, I158A, F181L, F186S, S187P, F191L.

[0048] A mutation that causes the complete loss of Fnr activity is particularly favored. This can be achieved either through specific amino acid substitutions in the Fnr protein (e.g., C20S, C23G, C29G, C122A, or G96D), or through alteration or deletion of DNA segments essential for Anr expression (e.g., in the Anr promoter region), or through partial or complete deletion of the Fnr gene. Co 12404 / Reu

[0049] 11

[0050] According to the invention, the ORF, encoding a recombinant protein, is functionally linked to a type II signal sequence encoding a signal peptide necessary for the secretion of recombinant target proteins from the cytoplasm into the periplasm. This means that the 5' end of the ORF of the target protein to be produced is linked in-frame to the 3' end of a signal sequence for protein export. In principle, all signal sequences of type II secretion systems that allow translocation via the See or TAT apparatus are suitable for this purpose. Various signal sequences are described in the prior art, such as... B. the signal sequences of the following genes: phoA, ompA, pelB, ompF, ompT, lamB, malE, spy, Staphylococcal protein A, Stil and others (Choi and Lee, 2004, Appl. Microbiol. Biotechnol. 64, 625-635).

[0051] According to the invention, the signal sequence of the phoA or ompA gene of E. coli, or the signal sequence for a cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae M5al, or the sequence derived from this signal sequence disclosed in US 2008 / 076157 Al, is preferably used for the secretion of recombinant target proteins from the cytoplasm into the periplasm. Particularly preferred is the signal sequence for a CGTase from Klebsiella pneumoniae M5al disclosed in EP 0 448 093, which is specified in the present invention by SEQ ID No. 3, as well as the sequence derived therefrom by SEQ ID No. 4, which is also disclosed in US 2008 / 076157 Al.

[0052] In the final step of the process according to the invention, the recombinant protein is isolated. For this purpose, the fermentation medium is separated from the solid components of the fermentation mixture, which essentially comprise the cells. This can be done, for example, by centrifuging or filtering the entire fermentation mixture, which contains the cells and the medium. The solid components of the fermentation mixture are then separated.

[0053] The 12 sets, which essentially comprise the cells, are subsequently discarded, while the centrifugation supernatant or filtrate contains the desired secreted target protein.

[0054] A preferred method for the fermentative production of recombinant proteins is characterized in that the bacterial strain of Enterobacteriaceae is a strain of the species Escherichia coli. Particularly preferably, it is a non-pathogenic bacterium.

[0055] In a preferred embodiment, the process for the fermentative production of recombinant proteins is characterized by the fact that the bacterial strain has a “leaky” mutation.

[0056] A "leaky mutation" is defined as a mutation in genes encoding structural elements of the outer cell membrane or cell wall, selected from the group of omp genes, tol genes, excD gene, excC gene, Ipp gene, pal gene, env genes, and Iky genes, which leads to increased release of periplasmic proteins into the medium (Shokri et al., 2003, Appl. Microbiol. Biotechnol. 60, pp. 654-664). Preferably, it is a "leaky" mutation in the Ipp gene, and particularly preferably a mutation selected from the group of Ippl mutations, Ipp3 mutations, and Ipp deletion mutations. An Ippl mutation is a mutation in the Ipp gene that results in the substitution of the arginine residue at position 77 for a cysteine ​​residue; an Ipp3 mutation is a mutation in the Ipp gene that results in the substitution of the glycine residue at position 14 for an aspartic acid residue. These mutations are described in detail in US 2008 / 254511.The Ipp deletion mutation is preferably a deletion of at least one nucleotide in the Ipp gene itself or in the promoter region of the Ipp gene, which leads to cells exhibiting increased leakiness for periplasmic proteins.

[0057] For the purposes of the present invention, increased leakiness means that after fermentation of the cells, a Co 12404 / Reu

[0058] 13 higher concentration of periplasmic proteins, e.g. alkaline phosphatase, is present in the culture medium than in a fermentation of the original strain before introduction of the “leaky” mutation under the same conditions.

[0059] The use of strains that, in addition to the attenuating fnr mutation, also have a “leaky” mutation (e.g. in the Ipp gene) is preferred for protein secretion via type II for higher extracellular yield (see examples 2 and 3 with fnr / lpp double mutants compared to fnr mutants).

[0060] A preferred method for the fermentative production of recombinant proteins is characterized in that the bacterial strain has a maximum of 75%, particularly preferably a maximum of 50%, and particularly preferably a maximum of 20% of the Fnr activity of the same strain before modification of its Fnr activity. A particularly preferred method for the fermentative production of recombinant proteins is characterized in that the bacterial strain has no Fnr activity.

[0061] A preferred method for the fermentative production of recombinant proteins is characterized by the fact that the chromosomal fnr gene is deleted in the bacterial strain.

[0062] The open reading frame (ORF) is the region of DNA or RNA that lies between a start codon and a stop codon and codes for the amino acid sequence of a protein. The ORF is also referred to as the coding sequence (CDS) of a gene.

[0063] ORFs are surrounded by non-coding regions. Therefore, the term gene refers to the DNA segment that contains all the basic information for producing biologically active RNA.

[0064] A gene therefore contains not only the DNA segment from which a single-stranded RNA copy is produced by transcription, but also additional DNA segments that are involved in the regulation Co 12404 / Reu

[0065] Fourteen of these components are involved in this copying process. Since a gene contains at least one ORF, a gene also codes for at least one protein. In the ORFs, each base triplet of DNA codes for a specific amino acid or a stop signal.

[0066] Within the scope of the present invention, a microorganism strain with which the target protein can be produced by fermentation is referred to as the production strain. The production strain particularly preferably comprises at least one gene construct.

[0067] The term "a / the recombinant protein," used in the singular within the scope of this invention, can also refer to several different recombinant proteins. Preferably, it refers to 1 to 3 different recombinant proteins, and particularly preferably to 1 or 2 different recombinant proteins. The recombinant protein is also referred to as the target protein.

[0068] Preferably, the recombinant proteins are heterologous proteins. For the purposes of the present invention, a heterologous protein is understood to be a protein that does not belong to the proteome, i.e., the entire natural protein composition, of the bacterial strain.

[0069] Preferably, the heterologous protein is a eukaryotic protein, particularly preferably a protein containing one or more disulfide bridges or existing in its functional form as a dimer or multimer, i.e., that the protein has a quaternary structure and is composed of several identical (homologous) or non-identical (heterologous) subunits.

[0070] The most important heterologous protein classes include antibodies and their fragments, cytokines, growth factors, and protein kinases. (Co 12404 / Reu)

[0071] 15 sen, protein hormones, lipocalins, anticalins, enzymes, binding proteins and molecular scaffolds and derived proteins and pharmacologically active peptides. Examples of these protein classes include heavy-chain antibodies and their fragments (e.g., nanobodies), single-chain antibodies, interferons, interleukins, interleukin receptors, interleukin receptor antagonists, G-CSF, GM-CSF, M-CSF, leukemia inhibitors, stem cell growth factors, tumor necrosis factors, growth hormones, insulin-like growth factors, fibroblast growth factors, platelet-derived growth factors, transforming growth factors, hepatocyte growth factors, bone morphogenetic factors, nerve growth factors, brain-derived neurotrophic factors (BDNF), glial cell line-derived neurotrophic factors, angiogenesis inhibitors, tissue plasminogen activators, coagulation factors, trypsin inhibitors, elastase inhibitors, and complement components.Hypoxia-induced stress proteins, proto-oncogene products, transcription factors, virus-constitutive proteins, proinsulin, parathyroid hormone, prourokinase, erythropoietin, thrombopoietin, neurotrophin, protein C, glucocerebrosidase, superoxide dismutase, renin, lysozyme, P450, prochymosin, lipocortin, reptin, serum albumin, streptokinase, tenecteplase, CNTF and cyclodextrin-glycosyltransferases.

[0072] Examples of proteins derived from molecular scaffolds include evibodies (derived from CTLA-4), affibodies (from protein A of S. aureus), avimers (from the human A-domain family), transbodies (from transferrin), DARPins (from the ankyrin repeat protein), adnectin (from fibronectin III), peptide aptamers (from thioredoxin), microbodies (from microprotein), affilins (from ubiquitin), α-crystallin, charybdotoxin, tetranectin, the PDZ domain of the RAS-binding protein AF-6, and the Kunitz-type domain of protein inhibitors.

[0073] Antibodies are a particularly favored class of proteins consisting of multiple protein subunits. Especially Co 12404 / Reu

[0074] 16. The bacterial strain is therefore preferably characterized by the fact that the heterologous protein is an antibody or a fragment of an antibody. Antibodies are used extensively in research, diagnostics, and as therapeutics, so that particularly efficient and technically feasible production processes are necessary.

[0075] Functional Fab antibody fragments and full-length antibodies can also be produced extracellularly using the method according to the invention. Preferred full-length antibodies are antibodies of the IgG and IgM classes, in particular of the IgG class.

[0076] In the production of functional Fab antibody fragments, the cell must produce the corresponding fragment of the light chain (LC), which includes the VL and CL domains, and the heavy chain.

[0077] (HC), which comprises the domains VH and CHI, are synthesized simultaneously and then secreted into the periplasm and finally into the fermentation medium. Outside the cytoplasm, the two chains are then assembled to form the functional Fab fragment.

[0078] In the process according to the invention, the recombinant protein is produced in increased yield. Increased yield with respect to the recombinant protein means that the yield of recombinant protein released into the culture medium is at least 1.1 times, preferably at least 1.5 times, and particularly preferably at least 1.8 times higher compared to the yield of recombinant protein in the culture medium obtained according to the prior art using a wild-type bacterial strain containing a gene for the recombinant protein and / or a wild-type bacterial strain containing a gene for Co 12404 / Reu

[0079] 17 the recombinant protein and additionally expressing a protein to destabilize the bacterial cell envelope can be produced.

[0080] The production of the DNA molecule, which comprises an in-frame fusion of a signal sequence and the ORF of the recombinant target protein, is carried out using methods known to those skilled in the art. For example, the coding region of the target protein can first be amplified by PCR using oligonucleotides as primers and then linked at the N-terminus to the DNA molecule comprising the sequence of a signal peptide, which was generated analogously to the coding region of the target protein, using standard molecular biology techniques. This linkage results in an in-frame fusion, i.e., a continuous reading frame encompassing both the signal sequence and the coding region of the target protein. Alternatively, the entire DNA molecule, comprising both of the aforementioned functional regions, can be produced by gene synthesis. This fusion can then be incorporated into a vector, e.g.,A plasmid, 3'-soapy, is introduced into a suitable promoter, which is then introduced into the cell via transformation, or directly integrated into the cell's chromosome using known methods. Preferably, the fusion of the signal sequence and the ORF encoding the recombinant protein is introduced into a plasmid, and the cell is transformed with this plasmid.

[0081] For the secretion of a recombinant target protein consisting of several different subunits from the cytoplasm into the periplasm, it is necessary to functionally link the ORFs of the genes of all subunits to be produced (target proteins) to a signal sequence for protein export. The ORFs of the genes of the different subunits can be linked to the same or different signal sequences. Linkage to different signal sequences is preferred; Co 12404 / Reu is particularly preferred.

[0082] 18 the linking of a subunit with the signal sequence of the phoA or ompA gene of E. coli and the linking of another subunit with the signal sequence for CGTase from Klebsi ella pneumoniae M5al with the sequence SEQ ID No. 3 or derived sequences such as the sequence SEQ ID No. 4.

[0083] The fusions of signal sequence and ORF encoding the respective subunits can then either be introduced into a vector, e.g., a plasmid, or integrated directly into the cell's chromosome using known methods. The individual subunits can be cloned with a signal sequence onto separate but compatible plasmids, or they can be cloned onto a single plasmid. The fusions can be combined into a single operon or expressed in separate cistrons. Combining them into a single operon is preferred. Similarly, the gene constructs can be combined into a single operon or integrated into the cell's chromosome in separate cistrons. Again, combining them into a single operon is preferred.

[0084] It is necessary that the DNA expression construct (fusion of signal sequence and ORF encoding the recombinant target protein) is equipped with functional expression signals in the chosen bacterial strain (promoter, transcription start site, translation start site, ribosome binding site, terminator).

[0085] All promoters known to those skilled in the art are suitable as promoters for the gene encoding the recombinant target protein, such as, on the one hand, inducible promoters like the lac, tac, trc, lambda PL, ara, or tet promoters, or sequences derived from them. On the other hand, constitutive expression can also be achieved by using a constitutive promoter.

[0086] 19 tors, such as the gapA promoter. However, the promoter normally linked to the gene of the recombinant protein to be produced can also be used.

[0087] This expression construct (promoter-signal sequence-sequence encoding the recombinant protein) is then introduced into cells of the respective production strain using methods known to those skilled in the art (e.g., transformation). The introduction of the expression construct for the production of the recombinant protein is carried out, for example, on a vector, such as a plasmid, such as a derivative of known expression vectors like pJF118EH, pKK223-3, pUC18, pBR322, pACYC184, pASK-IBA3, or pET. Suitable selection markers for plasmids are genes that encode resistance to, for example, ampicillin, tetracycline, chloramphenicol, kanamycin, or other antibiotics.

[0088] According to the invention, a bacterial strain is used in which the ORF is functionally linked to a type I signal sequence encoding a signal peptide active in the selected bacterial strain with functional expression signals in the selected bacterial strain, such as a promoter, a transcription and translation start site, a ribosome binding site and a terminator.

[0089] The cultivation (fermentation) of the bacterial cells takes place in a bioreactor (fermenter) according to standard fermentation methods known to experts.

[0090] The fermentation preferably takes place in a conventional bioreactor, for example a stirred tank reactor, a bubble column reactor, or an airlift reactor. A stirred tank reactor is particularly preferred. Co 12404 / Reu

[0091] 20

[0092] During fermentation, the cells of the protein-producing strain are cultivated in a liquid medium for a period of 16–150 hours, with various parameters such as nutrient supply, oxygen partial pressure, pH, and culture temperature being continuously monitored and precisely controlled. The cultivation period is preferably 24–72 hours.

[0093] In principle, all common media known to experts for the cultivation of microorganisms are suitable as culture media (fermentation media).

[0094] Complex media or minimal salt media, to which a defined proportion of complex components such as peptone, tryptone, yeast extract, molasses, or corn steep liquor is added, can be used as a medium for culturing bacterial cells. Chemically defined salt media, i.e., media with a precisely defined substrate composition (unlike complete media), are preferred for the production of pharmaceutical proteins. Examples of suitable minimal salt media for culturing bacterial cells, particularly E. coli cells, include the M9 minimal medium, the modified minimal medium, and the Riesenberg mineral medium (Kangwa et al., 2015, AMB Expr 5, p. 70), as well as the FM4 medium described in US 2008 / 0254511 Al.

[0095] In the process according to the invention, a bacterial strain exhibiting attenuated Fnr activity compared to an Anr wild-type strain, and containing an ORF encoding a recombinant protein which is linked in frame to a signal sequence encoding a type II signal peptide, grows to comparably high cell densities within a fermentation time comparable to that of a strain with Fnr wild-type protein and secretes the recombinant protein into the medium. Co 12404 / Reu

[0096] 21

[0097] In principle, any sugars, sugar alcohols, or organic acids or their salts that can be utilized by the cells can be used as the primary carbon source for fermentation. Glucose, lactose, or glycerol are preferred. Glucose and lactose are particularly preferred. A combined feeding of several different carbon sources is also possible. The carbon source can be completely introduced into the fermentation medium at the beginning of the fermentation, or none or only a portion of the carbon source can be introduced initially, with the carbon source being added during the course of the fermentation. A particularly preferred embodiment involves introducing part of the carbon source and adding part as it is fed.The carbon source is preferably presented at a concentration of 10-30 g / l, feeding is started when the concentration has dropped to less than 5 g / l and is designed to keep the concentration below 5 g / l.

[0098] The partial pressure of oxygen (pCt) in the culture is preferably between 10 and 70% saturation. A pCt between 20 and 60% is preferred, and a pÜ2 between 20 and 40% saturation is particularly preferred.

[0099] The pH of the culture is preferably between pH 6 and pH 8. Preferably, a pH between 6.5 and 7.5 is set, and particularly preferably, the pH of the culture is maintained between 6.8 and 7.2.

[0100] The culture temperature is preferably between 15 and 45 °C. A temperature range between 20 and 40 °C is preferred, a temperature range between 25 and 35 °C is particularly preferred, and 30 °C is most particularly preferred. Co 12404 / Reu

[0101] 22

[0102] A preferred method for the fermentative production of recombinant proteins is characterized in that the recombinant proteins are purified after isolation. As described above, isolation of the recombinant protein means that the solid components, essentially the bacterial cells, are separated from the fermentation medium and discarded. The recombinant proteins can then be purified from the fermentation medium. The purification of secreted proteins can be carried out by conventional purification methods known to those skilled in the art. The target protein can, for example, be concentrated by ultrafiltration and / or further purified by standard methods such as precipitation, chromatography, or ultrafiltration. Methods such as affinity chromatography are particularly preferred.The latter methods utilize the protein's already correctly folded native conformation.

[0103] A preferred method for the fermentative production of recombinant proteins is characterized in that, based on the total amount of recombinant protein produced, more than 50 wt.% and particularly preferably more than 55 wt.% are present in the medium.

[0104] Co 12404 / Reu

[0105] Examples

[0106] The following examples serve to further illustrate the invention without limiting it.

[0107] All molecular biological and microbiological procedures employed, such as polymerase chain reaction (PCR), gene synthesis, DNA isolation and purification, DNA modification by restriction enzymes, Klenow fragment and ligase, transformation, etc., were carried out in a manner known to those skilled in the art, described in the literature, or recommended by the respective manufacturers. The oligonucleotides used were obtained from Metabion International AG (Planegg, Germany).

[0108] Example 1: Generation of fnr deletion mutants of E. coli. The E. coli Anr wild-type strains JE5512 (HfrC man pps) (Hirota et al., 1977, Proc. Natl. Acad. Sei. USA 74, pp. 1417-1420, strain available from the National Institute of Genetics, Microbial Genetics Laboratory, NBRP E. coli, 1111 Yata, Mishima, Shizuoka, 411-8540 JAPAN) and the “leaky” strain JE5512 lpp3 (described in US 2021 / 301297 Al) were used as starting strains for the generation of .fnr deletion mutants.

[0109] In these strains, the coding region of the chromosomal .fnr wild-type gene (nucleotide 150-902 in SEQ ID No. 1) was almost completely removed using the X-Red recombinase system by Datsenko and Wanner (2000, Proc. Natl. Acad. Sci. USA 97, pp. 6640-6645). As a first step, the ORF of the fnr gene on the chromosome was replaced using this method with an expression cassette for an aminoglycoside O-phosphotransferase (gene bank no. EFB5119094), which conferred resistance to the antibiotic kanamycin (kanR) to the cells. In a second step, the resistance cassette, flanked by two homologous FRT sites, was removed using FLP recombinase from yeast, so that finally, instead of the ORF of Co 12404 / Reu

[0110] 24 fnr genes left a “scar” of approximately 100 base pairs including an FRT site in the chromosome.

[0111] The kanamycin resistance cassette was first amplified by PCR using the plasmid pKD4 (Coli Genetic Stock Center: CGSC #7632) as a template and the oligonucleotides fnrmut-fw (SEQ ID No. 5) and fnrmut-rev (SEQ ID No. 6). The first 60 nucleotides of fnrmut-fw are homologous to the start codon of the fnr open reading frame (ORF) and the sequence located 5' to it. The first 60 nucleotides of fnrmut-rev, on the other hand, are homologous to the stop codon of the bnr ORF and the sequence located 3' to it. This resulted in a linear DNA fragment of approximately 1,600 base pairs containing the kanamycin resistance cassette.

[0112] The two strains mentioned above were each transformed with the plasmid pKD46 (Coli Genetic Stock Center CGSC#: 7739). pKD46 contains the genes y, β, and exo under the expression control of an arabinose-inducible promoter. The gene products Garn, Bet, and Exo enable the recombination of a linear DNA fragment with the circular chromosomal DNA of the bacterium. In addition to an ampicillin resistance cassette, the pKD46 plasmid also contains a temperature-sensitive origin of replication, allowing the plasmid to be cured at elevated temperatures (approximately 37 °C to 42 °C).

[0113] Cells of JE5512 pKD46 and JE5512 lpp3 pKD46, containing the recombination-relevant proteins Garn, Bet, and Exo after growth in an arabinose-containing medium, were transformed with the linear DNA fragment containing the kanR cassette. Selection for integration of the kanR cassette into the chromosome of the strains at the position of the fnr wild-type ORF was performed on LB agar plates containing 50 mg / L kanamycin. This resulted in cells in which the fnr wild-type ORF had been completely replaced by the DNA fragment containing the kanR cassette. The correct chromosome integration was confirmed using Co 12404 / Reu.

[0114] 25

[0115] PCR using the oligonucleotides ogt-seq-fw (SEQ ID No. 7) and uspE-rev (SEQ ID No. 8) and chromosomal DNA of kanamycin-resistant cells as a template confirmed.

[0116] To remove the kanR gene from the chromosome, the cells were first cured of the pKD46 plasmid by incubation at 37 °C. The kanamycin-resistant cells were then transformed with the pCP20 plasmid (Coli Genetic Stock Center CGSC#: 7629), which, in addition to an ampicillin resistance cassette for selection and a temperature-sensitive origin of replication, also contains the gene for yeast FLP recombinase. Within the cells, the FLP recombinase interacts specifically with the two flanking FRT sites and ultimately eliminates the intervening kanR cassette, leaving one of the two FRT sites in the chromosome.

[0117] The cells were cured by incubation at 37 °C using the pCP20 plasmid. Successful deletion of the kanR gene from the chromosome was confirmed by PCR using the oligonucleotides ogt-seq-fw and uspE-rev(so) and chromosomal DNA from kanamycin-sensitive cells as a template. The resulting fnr deletion strains were designated JE5512 Afnr and JE5512 lpp3 Afnr, respectively.

[0118] Example 2: Fermentative production of a Fab-Antibody fragment with the Afnr mutants on a 3-1 scale

[0119] The present example describes the production of a Fab fragment of the humanized monoclonal anti-CD154 antibody 5c8, the sequence of which is published in Karpusas et al. (2001, Structure 9, pp. 321-329), using the fnr deletion mutants JE5512 Afnr and JE5512 lpp3 Afnr in comparison to the respective fnr wild-type strains JE5512 and JE5512 lpp3.

[0120] For the production of the anti-CD154 Fab antibody fragment on a fermenter scale, the strains JE5512, JE5512 lpp3, and JE5512 Co 12404 / Reu were used.

[0121] 26

[0122] Afnr and JE5512 lpp3 Afnr were transformed with the plasmid pJF118ut-CD154 using the CaC12 method. Selection for plasmid-containing cells was performed using tetracycline (20 mg / L).

[0123] The expression plasmid pJF118ut-CD154 (described in Coll805 = US 2021 / 301297 Al) contains, among other things, the genes for the heavy chain (VH-Chl domains) and for the light chain (VL-CL domains) of the Fab fragment, each including its own signal sequence, under the expression control of an IPTG-inducible promoter.

[0124] Production was carried out in 3 1 stirred tank fermenters. 1.2 1 of a mineral salt medium commonly used for cultivating E. coli, including 15 g / l glucose, enriched with complex components (1.5 g / l Hy-Express II (Kerry); 1.0 g / l Amisoy (Kerry); 0.5 g / l Hy-Yest (Kerry)), was inoculated to approximately an ODeoo = 0.01 with a pre-culture that had been cultivated in a shake flask for approximately 6 h in a complex medium (30 g / l Phytone Peptone (BD Biosciences); 5 g / l yeast extract (Oxoid); 5 g / l NaCl) at 30 °C. Inoculation represents point 0 of the fermentation process, or the start of fermentation. During fermentation, a temperature of 30 °C was maintained, and the pH was kept constant at 7.0 by adding NH4OH or H3PO4. The culture was stirred at 400 rpm at the beginning and aerated with 2 µg of compressed air purified through a sterile filter.Under these initial conditions, the oxygen probe was calibrated to 100% saturation prior to inoculation. The target value for Ct saturation during fermentation was set to 30%. After the O2 saturation dropped below the target value, a regulatory cascade was initiated to restore the Ct saturation to the target value. This involved first continuously increasing the gas supply to a maximum of 5 mV and then continuously increasing the stirring speed to a maximum of 1,500 rpm. Glucose feeding was started 10 hours after the start of cultivation. Approximately 0.5–1 hour before the planned induction, the temperature was lowered from 30 °C to 27 °C. Expression of Co 12404 / Reu was induced by adding isopropyl β-D-thiogalactopyranoside (IPTG) to 0.1 mM after approximately 21–23 hours of cultivation.

[0125] After 64 hours of cultivation, samples were taken, the cells were separated from the culture medium by centrifugation, and the Fab fragment content in the culture supernatant was determined using a sandwich ELISA test (su). To determine the amount of the target protein in the total culture broth, i.e., the sum of intracellular and extracellular Fab fragment, the Fab content in the homogenized culture broth was determined.

[0126] For this purpose, 150 pl of culture broth were mixed with 850 pl of 100 mM Tris / HCl buffer (pH 7.4) and the cells were lysis using a FastPrep homogenizer (FastPrep-24™ 5G, MP Biomedicals). After separation of the cell debris by centrifugation, the clear supernatant was used in the sandwich ELISA test.

[0127] The anti-CD154 Fab fragment was quantified using a sandwich ELISA assay known to those skilled in the art. An immobilized anti-Fd heavy chain antibody (The Binding Site, product number: PC075) served as the capture antibody, and a peroxidase-conjugated goat anti-human kappa light chain antibody (Sigma, product number: A7164) served as the detection antibody. Quantification was achieved by the conversion of the chromogenic substrate Dako TMB+ (Dako, product number: S1599) by the peroxidase and the resulting change in absorbance at 450 nm. The Fab fragment "Human Fab / Kappa" (Bethyl Laboratories, product number: P80-115) was used to calibrate the ELISA.

[0128] Table 1 lists the yields of the anti-CD154 antibody fragment in the culture supernatant and the total culture.

[0129] Table 1: Anti-CD154 titer in culture supernatant and total culture after 64 h fermentation Co 12404 / Reu

[0130] 28

[0131] Example 3: Fermentative production of a CYdodextrin-GlyCO-syl transferase with the Afnr mutants on a 3.1 scale

[0132] For the production of a cyclodextrin glycosyltransferase (CGTase) on a 3:1 scale, the strains JE5512, JE5512 Ipp3, JE5512 Afnr, and JE5512 ipp3 Afnr were transformed with the plasmid pCGT using the CaC12 method. Selection for plasmid-containing cells was performed using tetracycline (20 mg / L).

[0133] The production of the pCGT plasmid for CGTase overexpression is described in Example 4 of US 2008 / 0254511 Al, and the plasmid map is shown in Fig. 4 of US 2008 / 0254511 Al. Essentially, the plasmid contains, in addition to the gene for tetracycline resistance, the CGTase structural gene from Klebsiella pneumoniae M5al, including the native CGTase signal sequence. The expression of the CGTase-encoding gene is controlled by the tac promoter.

[0134] Cultivation for the fermentative production of CGTase with the strains JE5512 / pCGT, JE5512 Afnr / pCGT, JE5512 ipp3 / pCGT and JE5512 ipp3 Afnr / pCGT was carried out as described in Example 2. Co 12404 / Reu

[0135] 29

[0136] After 64 hours of fermentation, samples were taken and the CGTase content in the culture supernatant and the homogenized and clarified culture broth (see example 2) was subsequently determined by a CGTase activity test based on the amount of cyclodextrin (CD) enzymatically produced from starch.

[0137] CGTase activity test

[0138] Test buffer: 5 mM Tris / HCl buffer, 5 mM CaC12 x 2 H2O, pH 6.5

[0139] Substrate solution: 10% starch solution (Merck No. 1.01252) in test buffer, pH 6.5

[0140] Test setup: 0.2 ml substrate solution + 0.2 ml appropriately diluted CGTase sample (culture supernatant or homogenized and clarified culture broth)

[0141] Reaction temperature: 40°C

[0142] Enzyme test:

[0143] * Pre-tempering of substrate solution and CGTase-containing sample

[0144] (approx. 5 min at 40°C)

[0145] * Preparation of the test mixture by rapid mixing (whirl mixer) of substrate solution and CGTase-containing sample, diluting the sample with test buffer if necessary, so that a value of 0.9-1.5 g / 1 CD is determined in the subsequent HPLC analysis;

[0146] * Incubate for 3 minutes at 40°C

[0147] * Stop the enzyme reaction by adding 0.6 ml of methanol and mixing rapidly (whirl mixer)

[0148] * Cool the mixture on ice (approx. 5 min)

[0149] * Centrifuge (5 min, 12,000 rpm) and pipette off the clear supernatant

[0150] * Analysis of the quantity of CD produced by HPLC: The analysis was performed on an Agilent HP 1100 HPLC system with a Nucleodur 100-3 NH2-RP column (150 mm x 4.6 mm, Macherey-Nagel) and 64% acetonitrile in water (v / v) as the mobile phase, at a Co 12404 / Reu

[0151] A flow rate of 2.1 ml / min was used. Detection was performed using an RI detector (1260 Infinity RI, Agilent) and quantification was carried out based on peak area and an a-CD standard (Cavamax W6-8 Pharma, Wacker).

[0152] Calculation of enzyme activity: A = G*V1*V2 / (t*MG) [U / ml]

[0153] A = Activity,

[0154] G = CD content in mg / 1

[0155] VI = Dilution factor in the test mixture

[0156] V2 = Dilution factor of the CGT-containing sample before use in the test; if undiluted: V2 = 1 t = Reaction time in min

[0157] MG = Molecular weight in g / mol (MGCD = 973 g / mol)

[0158] 1 unit (U) = 1 pmol / 1 product (CD) / min

[0159] Table 2 shows the respective CGTase yields achieved.

[0160] Table 2: CGTase yield in the culture supernatant after 64 h fermentation

[0161] The proportion of CGTase released into the culture medium by the Afnr mutants relative to the total amount of enzyme produced by the cells was – as with the CD154 antibody fragment – ​​higher than in the corresponding Anr wild-type strain.

[0162] Examples 2 and 3 illustrate the fermentative production of a medically relevant Fab antibody fragment and a technical enzyme, respectively, using different E. coli strains.

[0163] In both cases, the Afnr- Co 12404 / Reu according to the invention are evident.

[0164] 31

[0165] Mutants are superior to their respective corresponding fnr wild-type strains with regard to the amount of target protein released into the culture medium.

Claims

Co 12404 / Reu 32 Patent claims 1. A method for the fermentative production of recombinant proteins characterized in that a bacterial strain from the family Enterobacteriaceae containing attenuated Fnr activity and containing at least one gene encoding a recombinant protein functionally linked to a type I I signal sequence encoding a signal peptide for transport into the periplasm is used, the recombinant protein is produced by these bacteria in a fermentative process and secreted into the medium, and the recombinant protein is isolated.

2. Method according to claim 1, characterized in that the bacterial strain of Enterobacteriaceae is a strain of the species Escherichia chia coli.

3. Method according to one or more of claims 1 to 2, characterized in that the attenuated Fnr activity is caused by the fact that the fnr gene is an allele of SEQ ID No. 1, wherein the sequence of the Anr allele is at least 80% identical to the sequence of SEQ ID No.

1.

4. Method according to one or more of claims 1 to 3, characterized in that the attenuated Fnr activity is caused by the presence of at least one mutation in the coding region of the fnr gene.

5. Method according to one or more of claims 1 to 4, characterized in that the attenuated Fnr activity Co 12404 / Reu is caused by a reduced expression of the fnr gene.

6. Method according to claim 4, characterized in that the coding region of the fnr gene is completely deleted.

7. Method according to claim 6, characterized in that the entire fnr gene is completely deleted.

8. Method according to one or more of claims 1 to 7, characterized in that the type II signal sequence is SEQ ID No. 3 or SEQ ID No.

4.

9. Method according to one or more of claims 1 to 8, characterized in that the bacterial strain has a “leaky” mutation.

10. Method according to one or more of claims 1 to 5 or 8 to 9, characterized in that the bacterial strain has a maximum of 75% of the Fnr activity of the same strain before modification of its Fnr activity.

11. Method according to one or more of claims 1 to 10, characterized in that the bacterial strain has no Fnr activity.

12. Method according to one or more of claims 1 to 11, characterized in that the chromosomal fnr gene is deleted in the bacterial strain.

13. Method according to one or more of claims 1 to 12, characterized in that the yield of recombinant protein released into the culture medium is at least 1.5 times higher compared to the yield of recombinant protein in the culture medium obtained with a wild-type Co 12404 / Reu 34 A bacterial strain containing a gene for the recombinant protein or a wild-type bacterial strain containing a gene for the recombinant protein and additionally expressing a protein to destabilize the bacterial cell wall can be produced.

14. Method according to one or more of claims 1 to 13, characterized in that the recombinant proteins are purified after isolation.

15. Method according to one or more of claims 1 to 14, characterized in that, with respect to the total amount of recombinant protein produced, more than 50 wt% is present in the medium.

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