Expression, refolding, and characterization of recombinant thrombopoietin/stem cell factor fusion protein in Escherichia coli.
Thrombopoietin/stem cell factor (TPO/SCF) is a novel fusion protein that combines the complementary biological effects of TPO and SCF into a single molecule. In this study, TPO/SCF gene was cloned into pET32a and expressed as a thioredoxin (Trx) fusion protein with a C-terminal 6His-tag in Escherichia coli BL21(DE3) under the control of T7 promoter. Trx-TPO/SCF protein approximately accounted for 20% of the total bacterial proteins and was found to accumulate in inclusion bodies. Inclusion bodies were separated from cellular debris, washed with buffer containing 2 M urea, and solubilized with 8 M urea. The refolding of Trx-TPO/SCF was then carried out by an on-column method. Soluble Trx-TPO/SCF was characterized for its dose-dependent effects on promoting cells proliferation in both TF1 and Mo7e cell lines. rhTPO/SCF was released by thrombin digestion and further purified by Ni(2+) affinity chromatography. Western blot analysis confirmed the identities of Trx-TPO/SCF and rhTPO/SCF.
Zang
Yuhui
Y
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, Nanjing, PR China.
Zhang
Xu
X
Jiang
Xiaoling
X
Li
Haoran
H
Zhu
Jie
J
Zhang
Chi
C
Peng
Wei
W
Qin
Junchuan
J
D015153:Blotting, Western; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000032:analysis / Q000302:isolation & purification; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000494:pharmacology; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D019089:Stem Cell Factor / Q000235:genetics* / Q000494:pharmacology; D013926:Thrombopoietin / Q000235:genetics* / Q000494:pharmacology
D029968:Escherichia coli Proteins; D036341:Intercellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; D019089:Stem Cell Factor; D013926:Thrombopoietin