OVERPRODUCTION OF SINGLE-CHAIN ANTIBODIES AGAINST HUMAN IFN-?2b IN Escherichia coli
AND ITS OBTAINING IN BIOLOGICALLY ACTIVE FORM
1Taras Shevchenko Kyiv National University, Ukraine;
2Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv;
The gene encoding mouse single chain antibody (ScFv) against human interferon ?2b (IFN??2b) was cloned into the plasmid vector under the control of promoter from phage T7 and the recombinant protein was expressed in Escherichia coli as inclusion bodies. After the isolation of inclusion bodies the desired protein containing affinity tail “6His tag” was solubilized and purified under denaturing conditions by immobilized-metal affinity chromatography. The soluble and purified ScFv was obtained by “on column” refolding and the recovery of biological activity were demonstrated. The higher levels of ScFv production for intracellular expression system in comparison with ScFv obtained by secretion were shown. The advantages of described refolding method are simplicity and high efficacy, moreover, refolding using a chromatographic process represents the manufacturable approach because it is easily automated using commercially available materials and preparative chromatography systems and also can be combined with simultaneous purification.
Key words: ScFv expression, inclusion bodies, refolding, protein purification.
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© The Ukrainian Biochemical Journal