Thioredoxin fusion/HIV-1 protease coexpression system for production of soluble human IL6 in E. coli cytoplasm

• Published in: Biochemistry and molecular biology international Vol. 46; no. 4; p. 839
• Main Authors: Han, B G, Ma, X K, Meng, L, Song, X G, Peng, S Y, Wang, J X, Ling, S G
• Format: Journal Article
• Language: English
• Published: England 01.11.1998
• Subjects: Amino Acid Sequence; Base Sequence; Cloning, Molecular; DNA Primers; Electrophoresis, Polyacrylamide Gel; Escherichia coli - genetics; HIV Protease - genetics; Humans; Interleukin-6 - genetics; Interleukin-6 - isolation & purification; Interleukin-6 - pharmacology; Molecular Sequence Data; Recombinant Fusion Proteins - genetics; Thioredoxins - genetics
• ISSN: 1039-9712
• DOI: 10.1080/15216549800204382

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.