Producing human mechano growth factor (MGF) in Escherichia coli.
MGF is a product of a unique muscle-specific splice variant of IGF1 gene (insulin-like growth factor). Its peculiar feature is a specific E-peptide, a 16 a.a. strand at the C-terminus. MGF increases cellular proliferation and inhibits terminal differentiation of myoblasts necessary for the secondary myotube formation. Previous analysis of physiological effects of MGF was performed using indirect methods such as RT-PCR based examination of the transcript contents in normal tissues, adenovirus-mediated DNA delivery and synthetic E-domain administration. Here, we describe isolation and purification of recombinant MGF thus allowing for the first time the possibility of direct examining MGF effects. The recombinant MGF of directly examining--was expressed in Escherichia coli as inclusion bodies (about 100-200mg/l), purified and refolded. Biological activity of refolded MGF was analyzed in vitro in proliferation assays with normal human myoblasts. As a result of our work, it has become possible to generate a standard MGF control with characterized activity and a ready-to use MGF test-system neither of which have been previously described. Our data open opportunities for the future works on MGF characterization and to the development of a powerful and highly specific therapeutic agent potentially applicable for muscle growth up-regulation, post-trauma muscle repair, age and hereditary myodystrophy mitigation and in sport medicine.
Kuznetsova
Tatiana V
TV
A.N. Bakh Institute of Biochemistry, Russian Academy of Sciences, Leninskii prosp., 33, Moscow 119071, Russia.
Schulga
Alexey A
AA
Wulfson
Andrey N
AN
Keruchenko
Jan S
JS
Ermolyuk
Yaroslav S
YS
Keruchenko
Irina D
ID
Tikhonov
Roman V
RV
Lisitskaya
Ksenia V
KV
Makarov
Andrey A
AA
Chobotova
Katya
K
Khomenkov
Vasily G
VG
Khotchenkov
Vyacheslav P
VP
Popov
Vladimir O
VO
Kirpichnikov
Mikhail P
MP
Shevelev
Alexei B
AB
D000595:Amino Acid Sequence; D001483:Base Sequence; D049109:Cell Proliferation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D032446:Myoblasts / Q000166:cytology; D010957:Plasmids; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D050799:STAT5 Transcription Factor / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
D011994:Recombinant Proteins; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; D025521:Tumor Suppressor Proteins