# US 2014/0154742 A1 · Novel expression and secretion vector systems for heterologous protein production in escherichia coli > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0154742 A1 - Document ID: US20140154742A1 - Status: Published application - Kind: A1 - Application number: 14/110,320 - Filed: 2012-04-08 - Published: 2014-06-05 - Earliest claimed: 2011-04-08 - Assignees: Anthem Biosciences Pvt Ltd. - Inventors: Ayyappan Nair; Sunil Kumar Sukumaran; Shalaka Samant; Gunja Gupta; Suthakarn Pichaimuthu; Ganesh Sambasivam - Web page: https://patentgrep.com/patent/US20140154742A1 ## CPC - C12N 15/70: Vectors or expression systems specially adapted for E. coli - C12N 15/625: {containing a sequence coding for a signal sequence} - C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione - C07K 2319/02: containing a signal sequence - C07K 2319/034: containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved - C07K 2319/035: containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract The present invention relates to a recombinant DNA expression/secretion system in E. coli wherein the said system combines the potential of signal peptide-based translocation of recombinant proteins to the periplasmic space of E. coli with membrane brave defective mutants of E. coli to further aid secretion into the extracellular space. The present invention further relates to the expression system which furthermore includes a helper plasmid to drive the expression of translocons to facilitate improved periplasmic secretion of the over-expressed recombinant protein. In addition, this system also facilitates efficient production of specific proteins of interest in E. coli. ## Claims ### Claim 1 A recombinant host cell comprising an expression vector, optionally along with a helper plasmid, wherein, the said recombinant cell is a membrane defective cell. ### Claim 2 (depends on 1) The expression vector as mentioned in claim 1 is capable of directing the expression and secretion of a protein, polypeptide or a peptide in a suitable host cell, wherein the expression vector further comprises a secretory signal sequence, inducible promoter and a gene of interest. ### Claim 3 (depends on 1) The helper plasmid as mentioned in claim 1 co-express translocons belonging to the SEC, TAT, SRP export pathway or in a combination thereof. ### Claim 4 (depends on 2) The expression vector as mentioned in claim 2 wherein gene of interest comprises a prokaryotic and eukaryotic gene. ### Claim 5 (depends on 2) The expression vector as mentioned in claim 2, wherein 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. ### Claim 6 (depends on 2) The expression vector as mentioned in claim 2, is used in a host cell wherein the host cell is a prokaryote. ### Claim 7 (depends on 6) The isolated host cell of claim 6, wherein said host cell is an Escherichia coli or a strain thereof. ### Claim 8 (depends on 2) The expression vector as mentioned in claim 2 comprises at least one of the plasmids selected from the group consisting of pAEV01, pAEV02, pAEV03, pAEV04, pAEV05, pAEV06 or pAEV07. ### Claim 9 A method of obtaining a recombinant cell, said method comprising steps of: a) obtaining a recombinant vector; b) transforming host cell with the recombinant vector; and c) optionally co-transforming the host cell with helper plasmid to obtain the recombinant cell. ### Claim 10 A method of obtaining recombinant peptide, method comprising steps of: a) obtaining recombinant vector comprising secretory signal sequence, inducible promoter and gene of interest; b) transforming host cell with the recombinant vector and optionally, co-transforming the host cell with helper plasmid; c) expressing the recombinant vector and secreting the recombinant peptide into extracellular medium; and d) optionally purifying the recombinant peptide. ### Claim 11 (depends on 9, 10) The helper plasmid as mentioned in claims 9 and 10 co-express translocons belonging to the SEC, TAT, SRP export pathway or in a combination thereof. ### Claim 12 (depends on 9, 10) The method of claims 9 and 10 wherein expression vector comprises at least one of the plasmids selected from the group consisting of pAEV01, pAEV02, pAEV03, pAEV04, pAEV05, pAEV06 or pAEV07. ### Claim 13 (depends on 9, 10) The method as mentioned in claims 9 and 10, wherein the expression vector comprises a codon optimized secretory 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, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. ### Claim 14 (depends on 10) The method as mentioned in claim 10 wherein the said inducible promoter is a T5 promoter. ### Claim 15 (depends on 7) The host cell as mentioned in claim 7 is having a defective outer membrane which is further co-transformed with the signal peptide-recombinant protein fusion vector and the translocon encoding plasmid. ### Claim 16 A kit for obtaining a recombinant peptide, said kit comprising an expression vector, a recombinant cell or a combination thereof.