Recombinant production of mecasermin in E. coli expression system.
Human Insulin-like growth factor 1 (hIGF-1) consists of 70 amino acids in a single chain with three intermolecular disulfide bridges possessing valuable therapeutic effects. To date, numerous variants of specifically engineered hIGF-1 have been produced so as to improve hIGF-1 biological activity, stability and stronger binding to IGF-1 receptor. Mecasermin is one of the modified variants with one amino acid substitution near the N-terminal (T4I) approved for the treatment of growth failure diabetes, wound healing, amyotrophic lateral sclerosis and severe primary IGF-1 deficiency. No scientific report for recombinant production of mecasermin in Escherichia coli (E. coli) expression system has been sofar reported. In the present study, we therefore investigated the overexpression of mecasermin in two different E. coli strains in order to obtain higher yield of recombinant protein. To achieve this goal, mecasermin DNA encoding sequence was designed based on polypeptide sequence, optimized according to E. coli codon preference, and cloned in pET15b. Recombinant vector, pET15-mecasermin, transferred into two E. coli strains rigami B (DE3) and BL21 (DE3) and induced for expression in a small scale. Results revealed the E. coli Origami B (DE3) expression system was a preferable host for mecasermin production due to its high expression level being around twice as much as BL21 (DE3). Large scale mecasermin production was performed in batch culture and produced recombinant protein specifically confirmed by western blotting and mass spectroscopy. Since major part of recombinant mecasermin was expressed as inclusion body, isolation and refolding was accomplished through developed purification procedure, and finally recombinant protein was successfully purified by gel filtration chromatography.
Department of Life Science Engineering, Faculty of New Science and Technologies, University of Tehran, I.R. Iran.
S
S
Jafari
Department of Life Science Engineering, Faculty of New Science and Technologies, University of Tehran, I.R. Iran ; Biochemistry Group, Biotechnology Research Center, Malek Ashtar University of Technology, Tehran, I.R. Iran.
V
V
Babaeipour
Department of Biochemistry, Isfahan Pharmaceutical Research Center and Bioinformatics Research Center, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.
H A Eslampanah
HA
Seyedi
Department of Life Science Engineering, Faculty of New Science and Technologies, University of Tehran, I.R. Iran.
M
M
Rahaie
Department of Biochemistry, Isfahan Pharmaceutical Research Center and Bioinformatics Research Center, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.
M R
MR
Mofid
Department of Biochemistry, Isfahan Pharmaceutical Research Center and Bioinformatics Research Center, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.
L
L
Haddad
Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Science, Tehran, I.R. Iran.
M M
MM
Namvaran
Genetic Engineering Group, Biotechnology Research Center, Malek Ashtar University of Technology, Tehran, Iran.
J
J
Fallah
Inclusion body; Insulin-like growth factor-1; Mecasermin; Over-expression; Purification; Western blotting