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Abstract

In this work we have successfully created two gene constructs pNanog and pOct4 in which target human genes Nanog and Oct4 were integrated under control of bacteriophage T7 promoter. After transformation of Rosetta (DE3) competent cells with vectors pNanog and pOct4 producer strains Rosetta (DE3)/pNanog and Rosetta (DE3)/pOct4 with confirmed high and stable expression of recombinant Nanog and Oct4 were obtained. It was found that the maximum synthesis of heterologous proteins in recombinant producer strains occurs within 2-4 hours of induction, which mainly are accumulated intracellularly in the form of inclusion bodies. Experiments on scaling up production of the recombinant Nanog and Oct4 showed that obtained bacterial strains can retain producing ability over 6 months. Moreover, biotechnological conditions for isolation and purification of recombinant Nanog and Oct4 from the insoluble fraction have been optimized. In particular, it was found that the isolation and purification of recombinant proteins is carried out by dissolving in 5 M urea supplemented with 10 mM 2-�²-mercaptoethanol followed by chromatography on Sepharose sorbent cross-linked with nickel ions. As a result, the yield of recombinant proteins Oct4 and Nanog was 2.9 and 6.5 mg from 1 liter of induced cultures for 4 hours and 16 hours respectively.

Biography

Vyacheslav Ogay has completed his PhD from Pushchino State University and Postdoctoral studies from Seoul National University School of Natural Sciences. He is the Head of Stem Cell Laboratory at National Center for Biotechnology. He has published more than 40 papers in peer reviewed journals on biotechnology and biomedicine.

Email: ogay@biocenter.kz