Thioredoxin fusion/HIV-1 protease coexpression system for production of soluble human IL6 in E. coli cytoplasm.
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.
Han
B G
BG
Institute of Basic Medical Sciences, Beijing, P. R. China.
Ma
X K
XK
Meng
L
L
Song
X G
XG
Peng
S Y
SY
Wang
J X
JX
Ling
S G
SG
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*
D017931:DNA Primers; D015850:Interleukin-6; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D016333:HIV Protease