title: Thioredoxin fusion/HIV-1 protease coexpression system for production of soluble human IL6 in E. coli cytoplasm.
abstract: In this paper, thioredoxin (TRX) fusion expression system has been modified to produce soluble human IL6 (hIL6) without TRX moiety in E. coli cytoplasm. A novel TRX gene fusion vector was developed that contained at the 3'-end of TRX gene a short DNA sequences encoding a linker peptide '-GSGSGVSQNYPIVQHHHHHH-', serving not only as a specific HIV-1 protease site but also providing six contiguous histidine (His) residues to foreign proteins. The cDNA for hIL6 was cloned into this vector resulting in plasmid pTRX@HISIL6. The cDNA for the HIV-1 protease has been cloned into another compatible plasmid pHMM2, resulting in plasmid pHMM2-PR. Both plasmids were transformed into E. coli strain GI724, and when induced for expression of both proteins, the correct processing of TRX@HISIL6 was obtained, producing hIL6 with His6-tag at the N terminus named HISIL6. A fraction of HISIL6 was found in soluble form and could be purified to homogeneity by Ni-NTA Superflow and ion-exchange chromatography. The biological activity of purified HISIL6 was measured by MTT method in an IL-6-dependent cell line 7TD1 to be 2.1 x 10(8) unit/mg.
authors.affiliation: Institute of Basic Medical Sciences, Beijing, P. R. China.
authors.forename: B G
authors.initials: BG
authors.lastname: Han
authors.forename: X K
authors.initials: XK
authors.lastname: Ma
authors.forename: L
authors.initials: L
authors.lastname: Meng
authors.forename: X G
authors.initials: XG
authors.lastname: Song
authors.forename: S Y
authors.initials: SY
authors.lastname: Peng
authors.forename: J X
authors.initials: JX
authors.lastname: Wang
authors.forename: S G
authors.initials: SG
authors.lastname: Ling
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D016333:HIV Protease / Q000235:genetics*; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D013879:Thioredoxins / Q000235:genetics*
chemical_list: D017931:DNA Primers; D015850:Interleukin-6; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D016333:HIV Protease