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A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells
Published on: July 31, 2012
Lab-scale Preparation of Recombinant Human Insulin-like Growth Factor-1 in Escherichia coli and its Potential Safety
Omnia A Mohamed1, Safia Samir1, Hanan Omar1
1Biochemistry and Molecular Biology Department, Theodor Bilharz Research Institute (TBRI), Giza, Egypt.
Background:
Insulin-like growth factor-1 (IGF-1) is structurally similar to insulin and acts as an endocrine hormone secreted by the liver.
Objective:
Production of recombinant human IGF-1 (rhIGF-1) in Escherichia coli (E.coli) and evaluation of its proliferation stimulatory activity.
Methods:
hIGF-1 gene cloned into pBSK (+) simple vector was transformed into TOP 10 chemically competent cells of E. coli. Polymerase chain reaction (PCR) was achieved using specific hIGF-1 gene primers to confirm the successful transformation. To express the rhIGF-1 in E. coli (Rosetta (DE3) pLysS); the hIGF-1 gene was cloned into the pET-15b expression vector and then the recombinant pET-15b/IGF-1 vector was transformed into a chemically prepared competent expression bacterial cells; Rosetta (DE3) pLysS. The rhIGF-1 was expressed as insoluble aggregates called inclusion bodies (IBs) using a 2 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) inducer. IBs were solubilized in a denatured form using 6 M guanidinium hydrochloride (GdmCl), followed by in vitro protein refolding using the rapid dilution method. The refolded hIGF-1 was purified using the HiTrap- ANX anion exchange column. Western blot and ELISA using rabbit polyvalent anti-hIGF- 1 were performed to confirm the protein antigenic identity. Cell proliferation activity of rhIGF-1 was testified on normal human lung cell line (WI-38).
Results:
rhIGF-1 was purified from the HiTrap-ANX column at a concentration of 300 μg/ml. Western blot showed a single 7.6 kDa band obtained in the induced Rosetta (DE3) pLYsS. ELISA confirmed the molecular identity of the rhIGF-1 epitope, the concentration of purified rhIGF-1 obtained from the ELISA standard curve using rhIGF-1 reference protein as a standard was 300 μg/ml, and activity on WI-38 cells was 2604.17I U/mg.
Conclusion:
Biologically active native rhIGF-1 protein was successfully expressed. Patents related to the preparation of IGF-1 were mentioned along the text.
