[Expression of mechano-growth factor in Escherichia coli and activity analysis].
Mechano-growth factor (MGF) is one of IGF-1 isoforms. MGF is mechanosensitive and has important functions in muscle hypertrophy, regeneration and nerve injury recovery. In this study, MGF cDNA (330 bp) was cloned from stretched osteoblasts by RT-PCR. In order to avoid prolin residue inhibiting enterokinase cleavage, 9bp of MGF cDNA 5' end sequence was truncated by primer, then the obtained truncated MGF (des(1-3)MGF) cDNA (321 bp) was subcloned in pET32a(+) vector to construct a prokaryotic recombination expression plasmid. Trx/des(1-3)MGF fusion protein, existing in forms of solution, was expressed in transformed Escherichia coli strain BL21(DE3) by IPTG induction at 30 degres C. The supernatant of cell lysates was subjected to ion exchange chromatography and Ni2+ metal affinity chromatography, and the fusion protein was obtained with the purity over 95%. After the fusion protein was cleaved by enterokinase, Trx and des(1-3)MGF was isolated by reverse-phase HPLC. Through these procedures, des(1-3) MGF was obtained with the purity of 98%. The protein molecular mass was conformity to the theoretical value by SDS-PAGE and mass spectrometry analysis. The purified des(1-3)MGF was incubated with MC3T3-E1 for cell proliferation and migration assays. The results show that des(1-3)MGF exhibited more facilitative effects on proliferation and migration of MC3T3-E1 than that of des(1-3)IGF-1.
Bioengineering College, Chongqing University, Chongqing 400030, China.
Bingbing
B
Zhang
Peng
P
Jiang
Chengyu
C
Xian
Yuxiao
Y
Li
Dajun
D
Li
Liling
L
Tang
Yuanliang
Y
Wang
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007334:Insulin-Like Growth Factor I; D010006:Osteoblasts / Q000378:metabolism; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D050799:STAT5 Transcription Factor / Q000096:biosynthesis* / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis* / Q000235:genetics
D018076:DNA, Complementary; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; D025521:Tumor Suppressor Proteins; D007334:Insulin-Like Growth Factor I