Compositions and methods for peptide expression and purification using a type iii secretion system

Patent metadata

Field	Value
Publication number	US-2018009851-A1
Application number	US-201715459191-A
Country	US
Kind code	A1
Filing date	Mar 15, 2017
Priority date	May 30, 2012
Publication date	Jan 11, 2018
Grant date	—

Abstract

Disclosed are compositions and methods for expressing and purifying a peptide of interest using a Flagellar Type III secretion system. Disclosed are nucleic acid sequences that contain a FlgM nucleic acid sequence, a cleavage site, and a nucleic acid sequence of interest. Also disclosed are polypeptides that contain FlgM, a cleavage site and a peptide of interest. Methods of producing polypeptides that have FlgM, a cleavage site and a peptide of interest are provided.

First claim

1 - 49 . (canceled)

50 . A recombinant cell line comprising one or more mutations in a gene selected from the group consisting of fliY, fliF, flgM, araBAD, fliA, fliC, fliD, prgH-hilA, cheV, tcp, yhjH, aer, mcpC, PmotA, motA-cheZ, flhDC, flgM, flgN, flgKL, trg, ycgR, mcpA, fliB, tsr, ecnR, hin-fljA, mcpB, lrhA, ydiV, flgE, and fljB;
wherein the recombinant cell line is a Salmonella enterica or an Escherichia coli cell line.

51 . The recombinant cell line of claim 50 , wherein the cell line is a strain selected from the group consisting of TH2788, TH4885, TH1539, TH10874, TH15360, TH15705, TH15706, TH15707, TH16229, TH16240, TH16778, TH17020, EM170, EM171, EM172, EM173, EM174, EM175, EM176, EM177, EM178, and EM179.

52 . The recombinant cell line of claim 50 , further comprising a vector comprising a FlgM nucleic acid sequence operably linked to a nucleic acid sequence encoding a purification tag, a cleavage site, a nucleic acid sequence of interest, and a transcription control element (TCE);
wherein the TCE is heterologous to the FlgM nucleic acid sequence; and wherein the 5′ to 3′ order of the sequences is the FlgM nucleic acid sequence, the nucleic acid sequence encoding a purification tag, the cleavage site, and the nucleic acid sequence of interest.

53 . The recombinant cell line of claim 50 , wherein the one or more mutations is in a coding region of the gene.

54 . The recombinant cell line of claim 50 , wherein the one or more mutations is in a ribosome binding sequence (RBS) of the gene.

55 . The recombinant cell line of claim 50 , wherein the one or more mutations is in a promoter of the gene.

56 . A recombinant cell line of claim 50 , wherein the one or more mutations is a mutation in fliA.

57 . The recombinant cell line of claim 56 , wherein the one or more fliA mutations is selected from the group consisting of V33E; L199R H14N; H14D; T138I; E203D; R91C; L207P; and a fliA start codon mutation from GTG to ATG.

58 . The recombinant cell line of claim 57 , wherein the one or more fliA mutation is selected from the group consisting of:
(a) a double mutant of R91C and L207P; (b) a triple mutant of H14D, R91C, and L207P; (c) a double mutant of fliA start codon change from GTG to ATG and H14N; and (d) a double mutant of fliA start codon change from GTG to ATG and a mutation in the fliA ribosome binding sequence (RBS) to the canonical sequence (CRBS).

59 . The recombinant cell line of claim 50 , wherein the background strain is derived from a fljBenx Vh2 mutant strain.

60 . The recombinant cell line of claim 52 , wherein the purification tag comprises poly-histidine, glutathione S-transferase (GST), Myc, HA, FLAG, or maltose binding protein (MBP).

61 . The recombinant cell line of claim 52 , wherein the FlgM nucleic acid sequence is wild type FlgM.

62 . The recombinant cell line of claim 52 , wherein the cleavage site comprises a Tobacco Etch Virus (TEV) protease cleavage site or an Enterokinase (ETK) cleavage site.

63 . The recombinant cell line of claim 52 , wherein the nucleic acid sequence of interest encodes a cysteine-rich peptide.

64 . The recombinant cell line of claim 63 , wherein the cysteine-rich peptide comprises a neuroactive toxin.

65 . The recombinant cell line of claim 52 , wherein the TCE is a constitutive TCE or a regulatable TCE.

66 . The recombinant cell line of claim 65 , wherein the regulatable TCE comprises an inducible promoter.

67 . The recombinant cell line claim 66 , wherein the inducible promoter comprises a P araBAD promoter.

68 . A FlgM peptide produced by the recombinant cell line of claim 52 .

69 . The FlgM peptide of claim 68 , wherein the purification tag comprises poly-histidine.

70 . A method of producing a peptide of interest comprising culturing the recombinant cell line of claim 52 in culture media.

71 . The method of claim 70 , wherein the bacterial host cell is cultured in media comprising about 200 mM to about 400 mM NaCl or KCl and wherein more of the peptide of interest is produced as compared to bacterial host cells cultured in media comprising 100 mM NaCl or KCl, respectively.

Assignees

• Univ Utah Res Found

Inventors

• Hughes Kelly T
• Olivera Baldomero M

Classifications

• C07K14/43518

  from spiders · CPC title

• C07K14/245 Primary

  Escherichia (G) · CPC title

• C07K14/46

  from vertebrates · CPC title

• C07K14/43522

  from scorpions · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title