JP2014512814A - A novel expression and secretion vector system for heterologous protein production in E. coli - Google Patents

Abstract

  The present invention relates to a recombinant DNA expression / secretion system in E. coli, said system further signaling the recombinant protein to the periplasmic space of E. coli to further aid secretion into the extracellular space. The ability of peptide-based translocation is combined with an incomplete membrane mutant of E. coli. The present invention further relates to an expression system further comprising a helper plasmid for driving the expression of the translocon in order to promote improved periplasmic secretion of overexpressed recombinant proteins. In addition, this system also facilitates efficient production of specific proteins of interest in E. coli.

Description

  The present invention relates to a recombinant DNA expression / secretion system in Gram negative prokaryotes such as, but not limited to, E. coli BL21 DE3 and E. coli K12. More particularly, the present invention provides the ability of a recombinant protein signal peptide-based translocation to the periplasmic space of E. coli to incomplete membrane mutants of E. coli to further facilitate secretion into the extracellular space. It relates to the system to be combined.

  Prokaryotes are widely used for the production of recombinant proteins. Controlled expression of the desired polypeptide or protein is achieved by linking the gene encoding the protein after the promoter through recombinant DNA technology, and the activity of the promoter can be regulated by external factors. This expression construct is carried in a vector, most often a plasmid. Introduction of a plasmid carrying the expression construct into the host bacterium and culturing the organism in the presence of a compound that activates the promoter results in high levels of expression of the desired protein. In this way, a large amount of the desired protein can be produced.

  E. coli is the most commonly used prokaryote for protein production. Many different types of plasmid vectors have been developed for use in E. coli to construct expression systems. Different variants use several different types of promoters, selectable markers and origins of replication, where each different configuration confers specific properties on the expression vector. In the most common arrangement, the expressed protein accumulates in the cytoplasm. This approach is useful for some proteins, but not all proteins can be accumulated in the cytoplasm in an active state. Often, if the desired protein is produced at a high level relative to the host protein, the protein accumulates as insoluble particles, also known as inclusion bodies. Proteins that accumulate as inclusion bodies are difficult to recover in active form.

  Two ways to solve this problem are either to send the target protein to the periplasm between the inner and outer membranes or to promote secretion into the extracellular space. Secreting the clonal gene product into the periplasmic space / extracellular medium has several potential advantages, including: 1) Protein products can circumvent cytoplasmic proteases; 2) Hormones Normally secreted proteins, such as lignin lytic enzymes and dextranases, can only fold in their active conformation in E. coli when secreted; 3) the periplasmic space has a more oxidative environment than the cytoplasm Can facilitate the correct formation of disulfide bonds; 4) toxic enzymes such as nucleases or proteases cannot be generated in the cytoplasm because of their ability to exert toxic effects on the host; and 5) Ease of purification.

  To target the periplasm to proteins, they are expressed as fusions with signal peptide sequences (eg PelB, OmpA, DsbA, TorA and MalE) that follow different secretory pathways (eg Sec, Tat and SRP). Other strategies include: a. Modification of the signal peptide to enhance translocation. b. Use of heterologous signal sequences in E. coli. c. Co-expression of periplasmic chaperones (eg Dsb family proteins). d. Protease negative mutant to reduce proteolysis.

  In many cases, delivery of the recombinant protein to the periplasm of E. coli is preferred for cytoplasmic production. However, if the protein is overexpressed, the delivery efficiency is significantly reduced and some of the benefits of the system are lost. In order to avoid overloading the host translocation mechanism following overexpression of the signal peptide-recombinant protein fusion, attempts were made to add the corresponding translocon from the secretory pathway to the original secretory mechanism.

  Extracellular production of recombinant proteins has several advantages over secretion into the periplasm. Extracellular production does not require disruption of the outer membrane to recover the target protein, thus it avoids intracellular proteolysis by periplasmic proteases and allows continuous production of recombinant proteins.

  Several methods have been applied to promote extracellular secretion of recombinant proteins from E. coli. These include the use of biochemicals, physical methods (osmotic shock, freezing and thawing), lysozyme treatment and chloroform shock. However, these methods can only be applied after the cells are collected. E. coli does not normally secrete proteins outside the cell, except for a few types of proteins such as toxins and hemolysin. In general, the transfer of recombinant proteins from the periplasm to the medium is the result of compromising the integrity of the outer membrane.

  Impairing the integrity of the bacterial outer membrane can be achieved by several approaches. One method involves fusing the product with a carrier protein (eg, hemolysin) that is normally secreted into the medium, or a protein (eg, OmpF) expressed on the outer membrane.

  Proteins secreted into the E. coli periplasm are released into the medium by co-expression of the gene encoding the third topology domain of kil or the transmembrane protein TolA (TolAIII) or the out gene from E. chrysanthemi EC16 You can also. Bacteriocin-releasing protein (BRP) can also be used for extracellular production of recombinant proteins in E. coli.

  Another approach for extracellular production of target proteins uses L-type bacterial cells, cells with no or imperfect walls. To construct the outer membrane protein (omp, tol, lpp, env) knockout and to promote the expression of the secreted recombinant protein, the corresponding leaking strain was used.

  Since E. coli K12 is a GRAS organism, it is a safe system for large-scale production of therapeutic proteins. However, K12 is not normally considered a secreted recombinant protein expression strain. The efficient secretory E. coli strain K12 generation approach described in this invention combines the ability of signal peptide-protein fusions and overexpression of translocon components with membrane incomplete mutants.

  The present invention takes advantage of the power of a novel signal peptide, and the nucleotide sequence of this peptide is selected so that the protein of interest can be directed to different cell compartments of E. coli cells. Thus, the present invention provides a platform of seven different signal peptides that can be tested to determine the best possible combination for secretory expression of the protein of interest.

  The present invention relates to recombinant DNA expression / secretion systems in Gram negative prokaryotes such as, but not limited to, E. coli K12 or E. coli BL21 DE3. The system combines the ability of a recombinant protein signal peptide-based translocation into the periplasmic space of E. coli with membrane incomplete mutants to further aid secretion into the extracellular space.

  Another aspect of the invention is an expression vector optionally comprising a helper plasmid that promotes translocon expression in order to promote enhanced periplasmic secretion of overexpressed recombinant protein. This system can be further used for the production of specific proteins secreted by E. coli hosts that are not normally secreted by this host. In addition, this system also facilitates efficient production of specific proteins of interest in E. coli. The expression vector contains a secretory signal sequence, an inducible promoter and the gene of interest.

  Yet another aspect of the present invention is a method for obtaining a recombinant cell, the method comprising: (a) obtaining a recombinant vector; (b) transforming a host cell with the recombinant vector; And (c) optionally co-transforming the host cell with a helper plasmid to obtain a recombinant cell.

  Yet another aspect of the present invention is a method for obtaining a recombinant peptide comprising the steps of: (a) obtaining a recombinant vector comprising a secretory signal sequence, an inducible promoter and a gene of interest; b) transforming the host cell with the recombinant vector and optionally co-transforming the host cell with a helper plasmid; (c) expressing the recombinant vector and placing the recombinant peptide in the extracellular medium; Secreting, and (d) optionally purifying to obtain a recombinant peptide.

  Yet another aspect of the invention includes a kit for obtaining a recombinant peptide comprising an expression vector, a recombinant cell or a combination thereof; and combining the expression vector, the recombinant cell or a combination thereof, A method for assembling a kit for obtaining a recombinant peptide.

  Reference will now be made to the exemplary embodiments illustrated in the accompanying drawings so that the present invention may be readily understood and practiced. The drawings are incorporated into and form a part of the specification together with the following detailed description to further illustrate embodiments according to the present invention and to illustrate various principles and advantages.

FIG. 4 is a schematic diagram representing a plasmid map of expression vector pAEV01 carrying a signal peptide-target protein fusion under the control of an inducible T5 promoter, in accordance with an embodiment of the present invention. FIG. 2 is a schematic diagram of the T7 promoter, signal peptide sequence, ribosome binding site and transcription start site of a Bacillus stearothermophilus maltogenic amylase according to an embodiment of the present invention. FIG. 6 shows SDS-PAGE analysis of lysates from strains expressing MalE, TorA and pelB signal peptide fusions with maltogenic amylase after induction of 0.1 mM IPTG, according to embodiments of the present invention. FIG. 7 shows an SDS-PAGE analysis of lysates from strains expressing DsbA, YcdO, FhuD, MdoD and pelB signal peptide fusions with maltogenic amylase after a 0.1 mM IPTG induction test according to embodiments of the present invention. is there. FIG. 4 shows SDS-PAGE analysis of lysates from strains expressing MalE, YcdO, TorA, FhuD and pelB signal peptide fusions with maltogenic amylase after a 1 mM IPTG induction test according to embodiments of the present invention. . FIG. 4 shows the induction factor due to 0.1 mM maltogenic amylase activity fused to pelB, MalE, FhuD, DsbA and MdoD compared to the corresponding non-induced cultures according to an embodiment of the present invention. FIG. 4 shows the induction factor due to 1 mM maltogenic amylase activity fused to MalE, FhuD, TorA, YcdO and pelB compared to the corresponding non-induced culture according to an embodiment of the present invention.

BRIEF DESCRIPTION OF THE TABLES Table 1 lists the amino acid residues of signal sequences and their delivery routes according to embodiments of the present invention.

  Table 2 lists signal sequences according to embodiments of the present invention.

  Table 3 lists signal sequence plasmid information according to embodiments of the present invention.

  In order to more clearly and concisely describe and illustrate the claimed subject matter, definitions for specific terms used in the following specification are set forth below.

  The term “expression” means transcription or translation, or both, depending on the context.

  The term `` expression vector '' refers to appropriate regulatory nucleotide sequences (e.g., promoters, enhancers, repressors, operator sequences and ribosome binding sites) that are necessary for the expression of operably linked nucleotide sequences in a particular host cell. Refers to a recombinant DNA molecule containing An expression vector may be self-replicating like a plasmid and thus carry a replication site, or it may be a vector that is integrated randomly into the host chromosome or at the target site. Expression vectors can contain a selection gene as a selectable marker that provides phenotypic selection in transformed cells. Expression vectors can also contain sequences that are useful for the control of translation.

  The term “operably linked / linked” or “operably linked” refers to initiating, modulating, or otherwise directing transcription and / or synthesis of a desired protein molecule relative to a regulatory nucleotide sequence. Refers to the located nucleotide sequence.

  The term “nucleotide” refers to a ribonucleotide or deoxyribonucleotide. “Nucleic acid” refers to a polymer of nucleotides and may be single-stranded or double-stranded. “Polynucleotide” refers to a nucleic acid of 12 or more nucleotides in length.

  The term “subject nucleotide sequence” means a nucleotide sequence that encodes a “subject protein, polypeptide or peptide sequence”, as those skilled in the art can consider that their production is desired for some reason. Such nucleotide sequences include, but are not limited to, coding sequences such as structural genes, regulatory genes, antibody genes, enzyme genes, or portions thereof. The nucleotide sequence of interest can include the coding sequence of a gene from one of many different organisms.

  A nucleotide sequence "encodes" or "codes for" a protein when the nucleotide sequence can be translated into the amino acid sequence of the protein. The nucleotide sequence may or may not include the actual translation initiation codon or termination codon.

  A “subject protein, polypeptide or peptide sequence” is encoded by a “subject nucleotide sequence”. A protein, polypeptide or peptide may be a protein from any organism, including but not limited to mammals, insects, microorganisms, eg, bacteria and viruses. It includes, but is not limited to, structural proteins, regulatory proteins, antibodies, enzymes, inhibitors, transporters, hormones, hydrophilic or hydrophobic proteins, monomers or dimers, therapeutic proteins, industrial proteins Or any type of protein, including a portion thereof.

  A “peptide” is a polymer of 4-20 amino acids, a “polypeptide” is a polymer of 21-50 amino acids, and a “protein” is a polymer of more than 50 amino acids.

  The term “portion” when used in reference to a protein refers to a fragment of that protein. Fragments can range from the size of the 4 amino acid residues of the protein to the size of the entire amino acid sequence minus one amino acid.

  The term “purified” or “purifying” refers to the removal of unwanted components from a sample. For example, purifying a secreted protein from a growth medium can mean removing other components of the medium (ie, proteins and other organic molecules), thereby increasing the percentage of secreted protein. As used herein, the terms “modified”, “mutant” or “variant” are used interchangeably and refer to: (a) one or more nucleotides are added or deleted, Or a nucleotide sequence substituted with a different nucleotide or modified base, or (b) a protein, peptide or polypeptide in which one or more amino acids have been added or deleted, or substituted with a different amino acid. Variants may be naturally occurring or may have been created experimentally by those skilled in the art. A variant may be a protein, peptide, polypeptide or polynucleotide that differs in one or more amino acids or nucleotides (ie, additions, deletions or substitutions) from the parent sequence.

  The term “periplasm” refers to the gel-like region between the outer surface of the cell membrane of Gram-negative bacteria and the inner surface of the lipopolysaccharide layer.

  The term “secretion” refers to the excretion of recombinant proteins expressed in bacteria to the periplasm or extracellular growth medium.

  According to a preferred embodiment, the invention relates to an expression vector comprising a secretory signal sequence, an inducible promoter and a gene of interest. The invention further relates to a recombinant cell, which is a membrane defective cell, optionally containing said vector together with a helper plasmid.

  Another preferred embodiment of the invention relates to a method of obtaining a recombinant cell, said method comprising the following steps: a. Obtaining a recombinant vector, b. Traiting a host cell with the recombinant vector. Transforming; and (c) co-transforming host cells, optionally with a helper plasmid, to obtain recombinant cells.

  Another embodiment relates to a method for obtaining a recombinant peptide, said method comprising the following steps: a. Obtaining a recombinant vector comprising a secretory signal sequence, an inducible promoter and a gene of interest; b. transforming the host cell with the recombinant vector and optionally co-transforming the host cell with a helper plasmid; c. expressing the recombinant vector and secreting the recombinant peptide into the extracellular medium And d. Optionally purifying to obtain a recombinant peptide.

  In an embodiment of the invention, the secretory signal sequence is a codon optimized sequence selected from the group comprising SEQ ID NO: 1 to SEQ ID NO: 7; the inducible promoter is a T5 promoter.

  In one embodiment of the invention, the gene of interest is selected from the group comprising prokaryotic and eukaryotic genes.

  In yet another embodiment of the invention, the cell is a prokaryotic cell, preferably E. coli K12; the helper plasmid is a plasmid carrying a chaperone or translocon from a prokaryotic secretion system.

  Another embodiment of the invention relates to a kit for obtaining a recombinant peptide comprising an expression vector, a recombinant cell or a combination thereof.

  The invention further includes a method of assembling a kit for obtaining a recombinant peptide comprising the act of combining expression vectors, recombinant cells or combinations thereof.

  In yet another embodiment of the invention, the helper plasmid is selected from the group comprising plasmids that carry any component of the chaperone or translocon from the bacterial secretion machinery; examples are SEC and TAT.

  The invention further relates to a method of producing a recombinant protein, polypeptide or peptide of interest through secretion of the recombinant protein, polypeptide or peptide of interest into the extracellular growth medium.

  In one embodiment of the invention, the method comprises a native E. coli secretory signal sequence to direct the secretion of recombinant protein, polypeptide or peptide to the periplasm through single or combined SEC, TAT or SRP delivery pathways. An expression vector carrying a specific codon-optimized variant of is utilized (Table 1).

  In an embodiment of the invention, the expression vector induces a signal sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. Carries downstream of possible T5 promoter (Table 2).

  In one embodiment, the expression vector is selected from the group consisting of plasmids pAEV01, pAEV02, pAEV03, pAEV04, pAEV05, pAEV06, pAEV07 (Table 3).

  In one embodiment, the expression vector is for use in a prokaryotic host cell, such as E. coli or a strain thereof.

  In yet another embodiment of the invention, an isolated that has been transformed by any of the expression vectors such that the cell expresses and secretes the protein, polypeptide or peptide of interest encoded by the nucleic acid. A host cell is provided. In one embodiment, the host cell is a prokaryotic host cell, such as E. coli or a strain thereof.

  In yet another embodiment of the present invention, the use of signal peptides corresponding to SEQ ID NOs: 2, 3 and 5 that utilize the characteristics of two periplasmic secretion signals in a single nucleotide sequence is described. Thus, the use of a corresponding expression vector can avoid blockage of specific secretion pathways and improve protein yield. The expression vectors described in the present invention carry signal peptide target protein fusions under the control of an inducible T5 promoter and thus adapt them for use in E. coli K12 hosts to induce heterologous protein expression.

  In yet another embodiment of the invention, the use of a helper plasmid to co-express translocons belonging to the SEC and TAT secretion pathways is described. Specifically, one of these helper plasmids encodes the genes secY, secE and secG as a single operon. Another helper plasmid encodes tatA, tatB and tatC as a single operon. Both these operons encoding the translocon are under the control of an inducible T5 promoter, thus adapting them for use in E. coli K12 hosts to induce heterologous protein expression.

  In yet another embodiment of the present disclosure, an E. coli strain having an incomplete outer membrane co-transformed with a signal peptide-recombinant protein fusion vector and a plasmid encoding a translocon. This E. coli strain not only targets the periplasmic space for recombinant proteins, but also facilitates passive diffusion of the leaked protein through the outer membrane into the extracellular medium.

(Example)
In order that the present invention may be more fully understood, the following preparatory and experimental examples are provided. These examples are for illustrative purposes only and should in no way be construed as limiting the scope of the invention.

(Example 1)
Cloning of the Maltogenic Amylase The following example shows the cloning of the maltogenic amylase-encoding gene into pET20b + and the seven other signal peptides described in the present invention SEQ ID NO: 1, 2, 3, 4, 5, 6 and 7 Illustrates the replacement of the pelB signal peptide of pET20b + maltogenic amylase (MA) by

  A maltogenic amylase gene from Bacillus stearothermophilus was amplified using primers carrying NcoI and BamHI sites. The primer sequences are as follows: MANcoI forward primer: 5'-gatcgtaccatgggaATGAGCAGTTCCGCAAGCGT-3 'and MABglII reverse primer: 5'-gatcgtacagatctTCTAGACTAGTTTTGCCACG-3'.

  The PCR product was then digested with NcoI and BglII enzymes and cloned into pET-20b (+) vector digested with NcoI and BamHI. The resulting ligation mixture was transformed into DH5α E. coli cells. The plasmid was verified for sequence to confirm the correct maltogenic amylase encoding gene and then digested with NdeI and NcoI to remove the pelB signal peptide by gel elution. Seven signal peptides (SEQ ID NOs: 1, 2, 3, 4, 5, 6 and 7) were synthesized with NdeI and NcoI overhangs and cloned into this vector. The resulting vector retained the reading frame defined by the ATG start codon from pET-20b (+) (FIG. 2). These seven plasmids were transformed into DH5α E. coli cells. The plasmid was isolated and verified from DH5α E. coli cells and then used to transform BL21 DE3 (a production strain capable of inducing heterologous gene expression using IPTG).

(Example 2)
This example illustrates induction testing with different maltogenic amylase signal peptide fusions using SDS-PAGE and a maltogenic amylase activity assay.

  All eight BL21 (DE3) strains were grown in minimal medium supplemented with glucose as a carbon source and 100 ug / ml ampicillin kept overnight in an incubator shaker at 37 ° C. and 200 rpm. The culture was diluted 1: 100, placed in a fresh 250 ml flask containing 50 ml yeast extract medium containing ampicillin and grown at 37 ° C. in a 200 rpm shaker incubator. When the OD at 600 nm reached 0.6, 0.1 mM IPTG was added to the culture. The culture was then incubated for 16 hours at 200 rpm and 26 ° C. Cultures induced and uninduced (grown in the same way as induced cultures except that no IPTG was added) were pelleted at 3500 rpm for 15 minutes. The pellet was resuspended in pH 5 sample buffer containing 10 mM NaCl. The pellet was sonicated to release soluble protein, cell debris was removed from the pellet, and the supernatant was analyzed by SDS-PAGE. Similarly, induction was performed by adding 1 mM IPTG to the culture and the induction temperature was maintained at 30 ° C.

  After induction with 0.1 mM IPTG and growth for 16 hours at 26 ° C., strains carrying the pAEV01, pAEV05, pAEV06 and pET-20b (+) constructs are the expected size of a maltogenic amylase on 12% SDS-PAGE A prominent protein band at 70 kDa was represented. Induction of maltogenic amylase protein levels with pAEV06 and pAEV01 was comparable to that of the parent vector, which was higher than that of the parent (FIGS. 3A and 3B). In strains carrying pAE04 and pAEV07, no maltogenic amylase was produced.

  The strain carrying the pAEV03 plasmid showed induction of the truncated protein, and the pAEV02 strain showed significant leaky expression of the maltogenic amylase, i.e. there was no difference in the amount of maltogenic amylase produced with or without induction. (FIGS. 3A and 3B).

  Experiments are performed to determine the localization of the maltogenic amylase protein. This elucidates the secretory nature of the signal peptide fusion. Our data suggest that fusion with different signal peptides contributes differently to the expression secretion of different proteins of interest.

  After induction with 1 mM IPTG and growth for 6 hours at 30 ° C., strains carrying the pAEV01, pAEV02, pAEV04, pAEV05 and pET-20b (+) constructs are at the expected size of maltogenic amylase on 12% SDS-PAGE. A prominent protein band at 70 kDa was represented (FIG. 4). This data shows that different IPTG concentrations and temperatures after induction play an important role in the expression of heterologous proteins.

  The sonicated supernatant was also subjected to the determination of maltogenic amylase activity using the glucose oxidase method. When cultures were induced with 0.1 mM IPTG, a higher fold induction was observed for maltogenic amylase activity with the pAEV01 construct compared to the parent (FIG. 5A).

  When the cultures were induced with 1 mM IPTG, both strains transformed with the pAEV05 and pAEV01 constructs showed a higher fold induction for maltogenic amylase activity compared to the parent (FIG. 5B). Strains carrying other constructs that showed maltogenic amylase expression on SDS-PAGE showed functional maltogenic amylase activity similar to the parent (FIGS. 5A and 5B).

  The results show that in the case of pAEV01 and pAEV05, a much higher amount of functional maltogenic amylase is directed to the periplasmic space compared to the parental plasmid. Regarding the localization of maltogenic amylase in the E. coli periplasmic gap, MalE and FhuD appear to represent an improved signal peptide compared to pelB.

  Thus, the present invention takes advantage of the power of a novel signal peptide that is optimized to assist in efficient translation of nucleotide sequences. These sequences are selected so that they can direct the protein of interest to different cell compartments of E. coli cells. Thus, the present invention provides a platform of seven different signal peptides that can be tested to determine the best possible combination for secretory expression of the protein of interest.

Claims (16)

  A recombinant host cell, which is a membrane-deficient cell, optionally containing an expression vector together with a helper plasmid.   2. The expression vector of claim 1 capable of directing the expression and secretion of a protein, polypeptide or peptide in a suitable host cell, further comprising a secretory signal sequence, an inducible promoter and the gene of interest.   2. The helper plasmid according to claim 1, wherein the helper plasmid coexpresses a translocon belonging to the SEC, TAT, SRP delivery pathway or a combination thereof.   3. The expression vector according to claim 2, wherein the gene of interest includes prokaryotic and eukaryotic genes.   The secretory signal sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. Item 3. The expression vector according to Item 2.   The expression vector according to claim 2, which is used in a prokaryotic host cell.   7. The isolated host cell according to claim 6, which is E. coli or a strain thereof.   The expression vector according to claim 2, comprising at least one plasmid selected from the group consisting of pAEV01, pAEV02, pAEV03, pAEV04, pAEV05, pAEV06 or pAEV07. a) obtaining a recombinant vector;
b) transforming a host cell with the recombinant vector;
c) optionally obtaining a recombinant cell comprising cotransforming the host cell with a helper plasmid to obtain a recombinant cell. a) obtaining a recombinant vector comprising a secretory signal sequence, an inducible promoter and the gene of interest;
b) transforming the host cell with the recombinant vector and optionally co-transforming the host cell with a helper plasmid;
c) expressing the recombinant vector and secreting the recombinant peptide into the extracellular medium;
d) optionally obtaining a recombinant peptide comprising the step of purifying the recombinant peptide.   The helper plasmid according to claim 9 and 10, wherein a translocon belonging to the SEC, TAT, SRP delivery pathway or a combination thereof is coexpressed.   11. The method according to claim 9 and 10, wherein the expression vector comprises at least one plasmid selected from the group consisting of pAEV01, pAEV02, pAEV03, pAEV04, pAEV05, pAEV06 or pAEV07.   Codon optimized secretion wherein the expression vector is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. 11. The method according to claim 9 and 10, comprising a sex signal sequence.   11. The method of claim 10, wherein the inducible promoter is a T5 promoter.   8. The host cell of claim 7, which has an incomplete outer membrane and is further co-transformed with a signal peptide-recombinant protein fusion vector and a plasmid encoding a translocon.   A kit for obtaining a recombinant peptide comprising an expression vector, a recombinant cell or a combination thereof.