KR20140004219A - Novel expression and secretion vector systems for heterologous protein production in escherichia coli - Google Patents

Abstract

The present invention relates to a recombinant DNA expression / secretion system in E. coli. This system combines the potential of signal peptide-based translocation of recombinant proteins with the E. coli membrane defect mutant to further secrete into the extracellular space. The present invention also relates to an expression system further comprising a helper plasmid that promotes expression of translocons to promote improved periplasmic secretion of the overexpressed recombinant protein. In addition, this system promotes the efficient production of specific proteins of interest in E. coli.

Description

Novel Expression and Secretion Vector Systems for Heterologous Protein Production in Escherichia Coli

The present invention relates to a recombinant DNA expression / secretion system in Gram-negative prokaryotic cells such as E. coli, including but not limited to E. coli BL21 DE3 and E. coli K12. More specifically, the present invention relates to a system that combines the potential of signal peptide-based translocation of recombinant proteins with membrane defective mutants of Escherichia coli to further aid secretion into the extracellular space.

Prokaryotic cells are widely used for the production of recombinant proteins. Controlled expression of the desired polypeptide or protein is achieved by binding the gene encoding the protein behind the promoter via recombinant DNA techniques, the activity of which can be controlled by external factors. This expression construct is carried on a vector, most often a plasmid. Injecting the plasmid carrying the expression construct into the host bacterium and culturing the organism in the presence of the compound activating the promoter results in high levels of expression of the desired protein. In this way, large amounts of the desired protein can be produced.

E. coli is the most commonly used prokaryotic cell for protein production. Several different variants of plasmid vectors have been developed for use in E. coli to create expression systems. Other variants use different types of promoters, selectable markers, and origins of replication, each of which gives different expressions unique characteristics. In the most common arrangement, expressed proteins accumulate in the cytoplasm. While this method is useful for some proteins, not all proteins can be active and accumulate in the cytoplasm. Primarily, when the desired protein is produced at a higher level than the host proteins, the protein accumulates in insoluble particles known as inclusion bodies. Proteins accumulated as inclusion bodies are difficult to remove in active form.

Two ways to solve this problem are to transport the target protein to the periplasm between the inner and outer membranes or to promote secretion into the extracellular space. There are several potential advantages in having a replication gene product secreted into the periplasmic space / extracellular medium, including: 1) the protein product can avoid cytoplasmic protease; 2) When secreted Normally secreted proteins such as hormones, lignin degrading enzymes and dextranases can only be folded into their active form in E. coli; 3) Accurate formation of disulfide bonds can be promoted because the surrounding cytoplasmic space provides a more oxidative environment than the cytoplasm; 4) Toxic enzymes such as nucleases or proteases cannot be produced in the cytoplasm because of their ability to exert a toxic effect on the host; And 5) ease of purification.

To target proteins to the periplasm, they are expressed in fusions with signal peptide sequences (eg, PelB, OmpA, DsbA, TorA, and MalE) along different secretory pathways (eg, Sec, Tat, and SRP). . Other strategies include a. Modification of Signal Peptides 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 strains that reduce proteolysis.

Transport of recombinant proteins to the periplasm of E. coli is desirable for cytoplasmic production in many cases. However, when the protein is overexpressed, the transport efficiency is significantly reduced and some benefits of the system disappear. Attempts have been made to supplement the intrinsic secretory machinery with corresponding translocons from the secretory pathways to avoid overloading the host's translocation machinery after overexpression of the signal peptide-recombinant protein fusion. lost.

Extracellular production of recombinant proteins has several advantages over secretion into the periplasm. Extracellular production does not require outer membrane breakdown to recover target proteins, thus avoiding intercellular proteolysis by periplasmic proteases and enabling continuous production of recombinant proteins.

Several methods have been used to facilitate the extracellular branching of recombinant proteins from E. coli. These methods include the use of biochemical, physical methods (osmotic shock, freezing and thawing), lysozyme treatment and chloroform shock. However, these methods can only be used after harvesting the cells. Escherichia coli generally do not secrete proteins extracellularly except for several kinds of proteins, such as toxins and hemolysine. In general, the transfer of recombinant proteins from the periplasm to the medium is a result of impaired integrity of the outer membrane.

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

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

Another approach to extracellular production of target proteins uses L-type cells, cells without walls or lack walls. Knockouts of outer membrane proteins (omp, tol, lpp, env) were prepared and corresponding leaky strains were used to promote secreted recombinant protein expression.

E. coli K12 is a GRAS organism that makes E. coli a safe system for the large-scale production of therapeutic proteins. However, K12 is generally not considered to be a secreted recombinant protein expression strain. One approach to the development of efficient secretory Escherichia coli K12 strains described herein combines the potential of overexpression of signal peptide-protein fusions and transrocon components with membrane deficient mutants.

The present invention uses the ability of new signal peptides to be selected such that the nucleotide sequence can direct the protein of interest to other cellular compartments of E. coli cells. On the other hand, 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 is to solve the above problem.

The present invention relates to a recombinant DNA expression / secretion system in Gram-negative prokaryotic cells such as E. coli, including but not limited to E. coli K12 or E. coli BL21 DE3. The system combines the potential of signal peptide-based translocation of recombinant proteins with the E. coli membrane defect mutant to further aid secretion into the extracellular space.

Another aspect of the invention is an expression vector optionally comprising a helper plasmid that promotes expression of translocons to promote improved periplasmic secretion of the overexpressed recombinant protein. This system can be further used for the production of specific proteins secreted by E. coli host, in which specific proteins are not normally secreted by the host. In addition, this system promotes the efficient production of specific proteins of interest in E. coli. Expression vectors include secretory signal sequences, inducible promoters and genes of interest.

Another aspect of the invention is a method of obtaining a recombinant cell, the method comprising (a) obtaining a recombinant vector, (b) transforming the host cell into a recombinant vector, and (c) optionally transforming the host cell into a helper plasmid. Comodifying to obtain recombinant cells.

Another aspect of the invention is a method of obtaining a recombinant peptide, the method comprising the steps of (a) obtaining a recombinant vector comprising a secretory signal sequence, an inducible promoter and a gene of interest, (b) transforming a host cell into a recombinant vector And optionally co-modifying the host cell with a helper plasmid, (c) expressing the recombinant vector and secreting the recombinant peptide into an extracellular medium, and (d) optionally purifying to obtain the recombinant peptide.

Another aspect of the invention is a kit for obtaining a recombinant peptide comprising an expression vector, a recombinant cell or a combination thereof and a method for assembling a kit for obtaining a recombinant peptide, the method comprising combining a recombinant vector, a recombinant cell or a combination thereof Steps.

Are included in the scope of the present invention.

Reference will now be made to the exemplary embodiments illustrated with reference to the accompanying drawings, in order that the present invention may be readily understood and substantially effective. The drawings, together with the description below, form a part thereof, further illustrate embodiments, and illustrate various principles and advantages in accordance with the present invention.
In accordance with embodiments of the present invention, FIG. 1 shows a schematic depiction of a plasmid map for an expression vector, pAEV01, carrying a signal peptide-target protein fusion under the control of an inducible T5 promoter.
In accordance with embodiments of the present invention, Figure 2 is a heat resistant bacterial maltose-producing amylase ( Bacillus) A schematic depiction of the T7 promoter, signal peptide sequence, ribosomal binding site and transcription start site of stearothermophilus maltogenic amylase.
In accordance with embodiments of the present invention, FIG. 3A depicts SDS-PAGE analysis of lysates from strains expressing MalE, TorA and pelB signal peptide fusions against maltose amylase following 0.1 mM IPTG induction.
In accordance with embodiments of the present invention, FIG. 3B shows SDS-PAGE analysis of lysates from strains expressing DsbA, YcdO, FhuD, MdoD and pelB signal peptide fusions to maltose amylase after 0.1 mM IPTG induction studies. Illustrated.
In accordance with embodiments of the present invention, FIG. 4 shows SDS-PAGE analysis of lysates from strains expressing MalE, YcdO, TorA, FhuD and pelB signal peptide fusions against maltose amylase after 0.1 mM IPTG induction studies. Illustrated.
In accordance with embodiments of the present invention, FIG. 5A shows fold induction by 0.1 mM maltose amylase activity fused to pelB, MalE, FhuD, DsbA and MdoD compared to the corresponding uninduced culture. do.
In accordance with embodiments of the present invention, FIG. 5B depicts fold induction by 1 mM maltose amylase activity fused to MalE, FhuD, TorA, YcdO and pelB compared to the corresponding uninduced culture.

In accordance with embodiments of the present invention, Table 1 lists the amino acid residues and individual transport pathways of the signal sequences.

In accordance with embodiments of the present invention, Table 2 lists the signal sequences.

In accordance with embodiments of the present invention, Table 3 lists signal sequence plasmid information.

In order to more clearly and briefly describe and point out the subject matter of the claimed invention, definitions are provided for specific terms used in the following description.

The term 'expression' means transcription or translation or both, as the content requires.

The term 'expression vector' refers to a recombinant DNA molecule comprising appropriate control nucleotide sequences (e.g., promoter, enhancer, repressor, operator sequence and ribosomal binding site) required for expression of the nucleotide sequence operably linked in a particular host cell. Means. Expression vectors can be autonomously replicated, such as plasmids, and therefore can be vectors that have a replication site or are integrated into the host chromosome at random or targeted sites. The expression vector may comprise a selection gene as a selectable marker for providing phenotypic selection in modified cells. The expression vector may comprise sequences useful for the control of translation.

The term 'operably linked / linked' or 'operably combined' refers to a nucleotide sequence positioned corresponding to control nucleotide sequences capable of initiating, regulating or directing the direct transcription and / or synthesis of a desired protein molecule. it means.

The term 'nucleotide' refers to ribonucleotides or deoxyribonucleotides. "Nucleic acid" means a polymer of nucleotides and may be single stranded or double stranded. "Polynucleotide" means a nucleic acid that is 12 or more nucleotides in length.

The term 'nucleotide sequence of interest' refers to a nucleotide sequence that encodes a 'protein of interest, polypeptide or peptide' which production would be desirable for whatever reason by those skilled in the art. Such nucleotide sequences include, but are not limited to, coding sequences of structural genes, regulatory genes, antibody genes, enzyme genes, or the like. The nucleotide sequence of interest may comprise a coding sequence of a gene from one of several different organs.

A nucleotide sequence 'encodes' or 'specifies genetic information' a protein if 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 start codon or end codon.

A protein of interest, polypeptide, or peptide sequence is encoded by a nucleotide sequence of interest. A protein, polypeptide or peptide can be a protein from any organism, including but not limited to microorganisms such as mammals, insects, bacteria and viruses. Proteins, polypeptides or peptides include, but are not limited to, structural proteins, regulatory proteins, antibodies, enzymes, inhibitors, transporters, hormones, hydrophilic or hydrophobic proteins, monomers or dimers, therapeutically related proteins, industrially related proteins, or portions thereof. It can be any form of protein, but not limited to.

A 'peptide' is a polymer of 4 to 20 amino acids, a 'polypeptide' is a polymer of 21 to 50 amino acids and a 'protein' is a polymer of at least 50 amino acids.

The term 'some' when used in connection with a protein refers to fragments of that protein. Fragments can vary in size from four amino acid residues of the protein to one amino acid from the entire amino acid sequence.

The term 'purified' or 'to purify' means the removal of unwanted components from a sample. For example, purifying the secreted protein from the growth medium means removing other components of the medium (ie, proteins and other organic molecules), thereby increasing the percentage of secreted protein. The terms 'modified', 'mutant' or 'variant' are used interchangeably herein and include (a) a nucleotide sequence in which one or more nucleotides have been added or deleted or substituted with other nucleotides or modified bases or (b) A protein, peptide or polypeptide wherein one or more amino acids are added, deleted or substituted with another amino acid. Variants can occur naturally or can be made experimentally by those skilled in the art. Variants may be proteins, peptides, polypeptides or polynucleotides that differ in one or more amino acids or nucleotides from the parent sequence (ie, additions, deletions or substitutions).

The term 'peripheral cytoplasm' refers to a gel-like region between the outer surface of the cytoplasmic membrane and the inner surface of the lipopolysaccharide layer of Gram-negative bacteria.

The term 'secretion' refers to the release of recombinant proteins expressed in bacteria into the periplasm or extracellular growth medium.

According to preferred embodiments, the present invention relates to an expression vector comprising a secretory signal sequence, an inducible promoter and a gene of interest. The invention also means a recombinant cell comprising said vector, optionally with a helper plasmid, wherein the recombinant cell is a membrane defective cell.

Another preferred embodiment of the invention relates to a method of obtaining recombinant cells, said method comprising a. Obtaining a recombinant vector, b. Transforming the host cell with a recombinant vector and c. Optionally co-modifying the host cell with a helper plasmid to obtain recombinant cells.

Another embodiment is a method of obtaining a recombinant peptide, the method comprising a. Obtaining a recombinant vector comprising a secretory signal sequence, an inducible promoter and a gene of interest, b. Modifying the host cell with a recombinant vector and optionally co-modifying the host cell with a helper plasmid, c. Expressing the recombinant vector and secreting the recombinant peptide into an extracellular medium, and d. Optionally purifying to obtain a recombinant peptide.

In one embodiment of the invention, said secretory signal sequence is a codon optimization sequence selected from the group comprising SEQ ID NO 1 to SEQ ID NO 7; Inducible promoters are T5 promoters.

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

In another embodiment of the invention, said cell is a prokaryotic cell, preferably E. coli K12; Helper plasmids are plasmids with chaperone or transrocon from prokaryotic secretion systems.

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

The invention also relates to a method of assembling a kit for obtaining a recombinant peptide, the method comprising the act of combining an expression vector, a recombinant cell or a combination thereof.

In another embodiment of the invention, the helper plasmid comprises a bacterial secretion apparatus; Exemplarily from SEC and TAT is selected from the group comprising plasmids with any component of chaperone or transrocon.

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

In one embodiment of the invention, the method comprises expression vectors with specific codon-optimized modifications of native E. coli secretory signal sequences to direct secretion of recombinant proteins, polypeptides or peptides via SEC, TAT or SRP transport pathways alone or in combination. (Table 1).

In one embodiment of the invention, the expression vector is 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; Has a signal sequence selected from the group consisting of downstream of the inducible 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 prokaryotic host cells, eg, E. coli or strains thereof.

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

In another embodiment of the present invention, sequence ID utilizing the characteristics of two periplasmic secretion signals in a single nucleotide sequence. No. The use of signal peptides corresponding to 2, 3 and 5 is described. Thus, the use of corresponding expression vectors can improve protein yield by avoiding blockage of certain secretory pathways. The expression vectors described herein will carry a signal-peptide target protein fusion under the control of an inducible T5 promoter and thus are suitable for use in an E. coli K12 host for inducing heterologous protein expression.

In another embodiment of the present invention, the use of a helper plasmid for coexpressing translocons belonging to SEC and TAT secretion pathways is described. In particular, one of these helper plasmids will encode the genes secY, secE and secG as a single operon. Another helper plasmid will encode tatA, tatB and tatC as a single operon. This transrocon, which encodes operons, will be under the control of an inducible T5 promoter and thus be suitable for use in an E. coli K12 host for inducing heterologous protein expression.

In another embodiment of the present invention, E. coli strains with defective outer membranes were co-modified with transrocons encoding signal peptide-recombinant protein fusion vectors and plasmids. Such E. coli strains will not only target the recombinant protein to the periplasmic space, but will also promote passive diffusion of leaky proteins across the outer membrane into the extracellular medium.

Example

In order that the present invention may be more fully understood, the following preliminary and experimental examples are described. These examples are illustrative only and are not to be construed as limiting the scope of the invention in any way.

Example  One

Maltose production Amylase  a copy

The following example clones the maltose amylase coding gene into pET20b + and the pET20b + maltose amylase (MA) by the seven other peptides SEQ ID NOs: 1, 2, 3, 4, 5, 6 and 7 described herein. The replacement of the pel B signal peptide is described.

Bacillus Maltose-producing amylase genes from stearothermophilus were amplified using primers with NcoI and BamHI sites. Primer sequences are MANcoI fwd primer: 5'-gatcgtaccatgggaATGAGCAGTTCCGCAAGCGT-3 'and MABglII rev primer: 5'-gatcgtacagatctTCTAGACTAGTTTTGCCACG-3'

This PCR product was then cleaved with NcoI and BalII enzymes and replicated into NcoI and BamHI cleaved pET-20b (+) vectors. The resulting ligation mix was modified with DH5α Escherichia coli cells. The plasmid was a sequence proven to identify the correct maltose amylase coding gene and was cleaved with NdeI and NcoI to remove the pelB signal peptide by gel elution. Seven signal peptides (SEQ. ID. No. 1, 2, 3, 4, 5, 6 and 7) were synthesized with NdeI and NcoI overhangs and replicated in this vector. The obtained vectors had a reading frame defined by ATG start codon from pET-20b (+) (FIG. 2). These seven plasmids were transformed into DH5α Escherichia coli cells. Plasmids were isolated from DH5α Escherichia coli cells and confirmed and used to modify BL21 DE3 (producer strains where heterologous gene expression can be induced using IPTG).

Example  2

This example describes induction studies for other maltose amylase signal peptide fusions using SDS-PAGE and maltose amylase activity assays.

All eight BL21 (DE3) strains were grown in minimal medium supplemented with glucose and 100 ug / ml ampicillin as the carbon feedstock and maintained overnight at 37 ° C., 200 rpm in a culture mixer. This culture was diluted 1: 100 in a fresh 250 ml flask with 50 ml yeast extract medium containing ampicillin and grown at 37 ° C. in a culture mixer at 200 rpm. 0.1 mM IPTG was added to the culture when the OD reached 0.6 at 600 nm. The culture was then incubated at 26 ° C. for 16 h at 200 rpm. Induced and non-induced cultures (growing in the same manner as induced cultures except that no IPTG was added) were pelleted down at 3500 rpm for 15 minutes. The pellet was resuspended in sample buffer containing 10 mM NaCl, pH 5. This pellet was sonicated to release soluble protein, the cell fragments were pelleted out and the supernatants analyzed on SDS-PAGE. Induction was similarly performed by adding 1 mM IPTG to the culture and the induction temperature was maintained at 30 ° C.

Strains with pAEV01, pAEV05, pAEV06, and pET-20b (+) constructs after induction by 0.1 mM IPTG and grown for 16 h at 26 ° C. had a prominent protein band at 70 kDa, the expected size of maltose amylase on 12% SDS-PAGE Indicated. Induction of maltose amylase protein levels in pAEV06 and pAEV01 was comparable to induction of parental vector and induction of pAEV05 was higher than parental vector (FIGS. 3A and 3B). There was no maltose amylase produced in strains with pAE04 and pAEV07.

Strains with the pAEV03 plasmid showed induction of the truncated protein and pAEV02 strain showed a marked leaky expression of maltose amylase, i.e. the difference in the amount of maltose amylase produced with and without induction None (FIGS. 3A and 3B).

Experiments will be conducted to determine the localization of the maltose-producing amylase protein. This will illuminate the secretion properties of the signal peptide fusions. The inventor's data suggest that fusions to other signal peptides will affect the expression secretion of different proteins of interest differently.

Strains with pAEV01, pAEV02, pAEV04, pAEV05, and pET-20b (+) constructs after induction by 1 mM IPTG and grown for 6 h at 30 ° C. showed significant protein at 70 kDa, expected size of maltose amylase on 12% SDS-PAGE The band is shown. This data indicates that different IPTG concentrations and post-induction temperatures play an important role in heterologous protein expression.

Maltose-producing amylase activity was measured on the sonicated supernatant using the glucose oxidase method. Higher fold induction in terms of maltose amylase activity was observed in the pAEV01 construct compared to the parent when cultures were induced with 0.1 mM IPTG (FIG. 5A).

Both pAEV05 and pAEV01 construct modified strains showed higher fold induction in terms of maltose-producing amylase activity when the cultures were induced with 1 mM IPTG (FIG. 5B). Strains with other constructs showing maltose amylase expression on SDS-PAGE exhibited functional maltose amylase activity similar to parent (FIGS. 5A and 5B).

The results indicate that in the case of pAEV01 and pAEV05 a much higher amount of functional maltose amylase is targeted into the periplasmic space compared to the parental plasmid. MalE and FhuD appear to show improved signal peptides compared to pel B for maltose amylase localization into the E. coli periplasmic space.

Signal sequence Route Amino acid sequence for each signal sequence Obtained plasmid MalE SEC MKIKTGARILALSALTTMMFSASALA pAEV01 YcdO TAT + SEC MTINFRRNALQLSVAALFSSAFMANA pAEV02 MdoD TAT + SEC MDRRRFIKGSMAMAAVCGTSGIASLFSQAAFA pAEV03 TorA TAT MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATAAQAA pAEV04 FhuD TAT + SEC MSGLPLISRRRLLTAMALSPLLWQMNTAHA pAEV05 DsbA SRP MKKIWLALAGLVLAFSASAAQ pAEV06 OmpA SEC MKKTAIAIAVALAGFATVAQA pAEV07

Seq ID no.1 atgaaaattaaaaccggcgcgcgcattctggcgctgagcgcgctgaccaccatgatgtttagcgctagcgcgctggcc
" MalE " Seq ID no.2 atgaccattaactttcgccgcaacgcgctgcagctgagcgtggcggcgctgtttagcagcgcgtttatggcgaacgcc " YcdO " Seq ID no.3 atggatcgccgccgctttattaaaggcagcatggcgatggcggcggtgtgcggcaccagcggcattgctagcctgtttagccaggcggcgtttgcc
" MdoD " Seq ID no.4 atgaacaacaacgatctgtttcaggcgagccgccgccgctttctggcgcagctgggcggcctgaccgtggcgggcatgctggggcccagcctgctgaccccgcgccgcgcgaccgcggcgcaggcc
" TorA " Seq ID no.5 atgagcggcctgccgctgattagccgccgccgcctgctgaccgcgatggcgctgagcccgctgctgtggcagatgaacaccgcgcatgcc
" FhuD " Seq ID no.6 atgaaaaaaatttggctggcgctggcgggcctggtgctggcgtttagcgctagcgcc
" DsbA " Seq ID no.7 atgaaaaaaaccgcgattgcgattgcggtggcgctggcgggctttgcgaccgtggcgcaggcc

" OmpA "

Plasmid
name Secretory pathway Full base pairs ( bp ) Signal sequence pAEV01 SEC 5,784 atgaaaattaaaaccggcgcgcgcattctggcgctgagcgcgctgaccaccatgatgtttagcgctagcgcgctggcc pAEV02 TAT + SEC 5,784 atgaccattaactttcgccgcaacgcgctgcagctgagcgtggcggcgctgtttagcagcgcgtttatggcgaacgcc pAEV03 TAT + SEC 5,802 atggatcgccgccgctttattaaaggcagcatggcgatggcggcggtgtgcggcaccagcggcattgctagcctgtttagccaggcggcgtttgcc pAEV04 TAT 5,832 atgaacaacaacgatctgtttcaggcgagccgccgccgctttctggcgcagctgggcggcctgaccgtggcgggcatgctggggcccagcctgctgaccccgcgccgcgcgaccgcggcgcaggcc pAEV05 TAT + SEC 5,796 atgagcggcctgccgctgattagccgccgccgcctgctgaccgcgatggcgctgagcccgctgctgtggcagatgaacaccgcgcatgcc pAEV06 SRP 5,763 atgaaaaaaatttggctggcgctggcgggcctggtgctggcgtttagcgctagcgcc pAEV07 SEC 5,769 atgaaaaaaaccgcgattgcgattgcggtggcgctggcgggctttgcgaccgtggcgcaggcc

Thus, the present invention exploits the ability of new signal peptides whose nucleotide sequences are optimized to support efficient translation. These sequences are selected to direct other cell compartments of E. coli cells. On the other hand, 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.

Sequence List

SEQUENCE LISTING <110> Anthem Biosciences Pvt Ltd <120> Novel expression and secretion vector systems for heterologous protein production in E. coli <160> 7 <210> 1 <211> 78 <212> DNA <213> <220> <400> 1 atgaaaatta aaaccggcgc gcgcattctg gcgctgagcg cgctgaccac catgatgttt 60 agcgctagcg cgctggcc 78 <210> 2 <211> 78 <212> DNA <213> <220> <400> 2 atgaccatta actttcgccg caacgcgctg cagctgagcg tggcggcgct gtttagcagc 60 gcgtttatgg cgaacgcc 78 <210> 3 <211> 96 <212> DNA <213> <220> <400> 3 atggatcgcc gccgctttat taaaggcagc atggcgatgg cggcggtgtg cggcaccagc 60 ggcattgcta gcctgtttag ccaggcggcg tttgcc 96 <210> 4 <211> 126 <212> DNA <213> <220> <400> 4 atgaacaaca acgatctgtt tcaggcgagc cgccgccgct ttctggcgca gctgggcggc 60 ctgaccgtgg cgggcatgct ggggcccagc ctgctgaccc cgcgccgcgc gaccgcggcg 120 caggcc 126 <210> 5 <211> 90 <212> DNA <213> <220> <400> 5 atgagcggcc tgccgctgat tagccgccgc cgcctgctga ccgcgatggc gctgagcccg 60 ctgctgtggc agatgaacac cgcgcatgcc 90 <210> 6 <211> 67 <212> DNA <213> <220> <400> 6 atgaaaaaaa tttggctggc gctggcgggc ctggtgctgg cgtttagcgc tagcgcc 57 <210> 7 <211> 63 <212> DNA <213> <220> <400> 7 atgaaaaaaa ccgcgattgc gattgcggtg gcgctggcgg gctttgcgac cgtggcgcag 60 gcc 63

Claims (16)

A recombinant host cell, optionally comprising a helper plasmid, comprising an expression vector, wherein said recombinant cell is a membrane defective cell. The method of claim 1,
An expression vector can direct the expression and secretion of a protein, polypeptide or peptide in a suitable host cell and further comprises a secretory signal sequence, an inducible promoter and a gene of interest. The method of claim 1,
The helper plasmid is a recombinant host cell that co-expresses a transrocon belonging to the SEC, TAT, SRP transport pathway, or a combination thereof. 3. The method of claim 2,
The recombinant host cell of which the gene of interest comprises prokaryotic and eukaryotic genes. 3. The method of claim 2,
Secretory signal sequences include 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 a recombinant host cell selected from the group consisting of SEQ ID NO: 9. 3. The method of claim 2,
The expression vector is used in a host cell which is a eukaryotic cell. The method according to claim 6,
The host cell is E. coli or a strain thereof. 3. The method of claim 2,
The expression vector comprises at least one of the plasmids selected from the group consisting of pAEV01, pAEV02, pAEV03, pAEV04, pAEV05, pAEV06 or pAEV07. a) obtaining a recombinant vector;
b) transforming the host cell into a recombinant vector; And
c) optionally co-modifying the host cell with a helper plasmid to obtain the recombinant cell. a) obtaining a recombinant vector comprising a secretory signal sequence, an inducible promoter and a gene of interest;
b) modifying the host cell with a recombinant vector and optionally comodifying the host cell with a helper plasmid;
c) expressing the recombinant vector and secreting the recombinant peptide into an extracellular medium; And
d) optionally purifying to obtain a recombinant peptide. 11. The method according to claim 9 or 10,
The helper plasmid is a coexpression of a transrocon belonging to the SEC, TAT, SRP transport pathway, or a combination thereof. 11. The method according to claim 9 or 10,
The expression vector comprises at least one of plasmids selected from the group consisting of pAEV01, pAEV02, pAEV03, pAEV04, pAEV05, pAEV06 or pAEV07. 11. The method according to claim 9 or 10,
The expression vector is 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 And a codon optimized secretory signal sequence selected from the group consisting of SEQ ID NO: 9. 11. The method of claim 10,
Said inducible promoter is a T5 promoter. The method of claim 7, wherein
The host cell is a recombinant host cell having a defective outer membrane co-modified further with a translocone encoding a signal peptide-recombinant protein fusion vector and a plasmid. A kit for obtaining a recombinant peptide comprising an expression vector, a recombinant cell or a combination thereof.

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