Isaura Patricia Torres G, Sinar David Granada G, Ana María Aristizábal O, Juan Sebastian Herrera, Yuri Maritza Zapata, Cesar Segura L, Carlos Uribe, Orville Hernández R, Ligia Luz Corrales G, Ana María García, C, Oscar Mauricio Gómez
Department of Cellular and Molecular Biology Laboratory, Corporación para Investigaciones Biológicas (CIB), Colombia
EGF is a 6.2 kDa peptide with cell proliferation activity, with cosmetic and medical applications at industrial level and recombinant microorganisms are the most cost effective way to produce it. We intend to obtain a recombinant Escherichia coli isolate producing hEGF using the InvitrogenTM Gateway® system.
A synthetic hEGF gene was cloned in pDEST17 and transformed into E. coli-BL21-AI. Characteristics of the vector and insert were confirmed by restriction analysis, PCR and sequencing. Effectiveness of hEGF induction with L-arabinose was confirmed by SDS-PAGE and Western blot. Quantification of the expression was made by Byuret and the characteristics of pure hEGF were evaluated by SDS-PAGE, Western blotting, HPLC, mass spectrometry and cell proliferation assays.
Clone 100-1 produces a 10 kD protein (hEGF-CIB-TQBL21-A1) coincident with hEGF but with an extra His-Tag sequence on inclusion bodies, corresponding to 70% of total protein and had an expression yield of 450 mg/L. SDSPAGE and RP-HPLC showed a purity >99%of the protein. The refolded active protein showed similar biological activity compare to standard hEGF (Roche).
A productive clone, E. coli-BL21-AI-hEGF-clon-100-1, was obtained. The protein hEGF- CIB-TQBL21-A1 was successfully purified. All its features, including were almost indistinguishable from control hEGF (Roche), confirming the biosimilarity of CIB-TQBL21-A1.
Funding: This project is sponsored and is part of the Biotechnology initiative of Laboratory Tecnoquímicas SA.
Keywords: EGF, cloning, E. coli, Gateway