Factors influencing the expression of NS1 protein from avian influenza virus H5N1 subtype

Objective To optimize the culture conditions for the high expression of NS1 protein.Methods Engineering bacteria were inoculated into LB medium and incubated with shaking.The IPTG concentration and culture temperature were set for 0.3mmol/L and 37℃ respectively,then the duration of induction were optimized.Inductions were initiated in turn at 0.5h intervals from 0.5h to 6.0h.The expression of fusion protein were assessed by SDS-PAGE using 12% acrylamide.The culture temperature and the duration of induction were set for 37℃ and 4.0h respectively,then the amount of IPTG were added from 0.1mmol/L to 1.0mmol/L final concentration.and the expression of fusion protein were assessed by SDS-PAGE.The IPTG concentration and the duration of induction were set for 0.6mmol/L and 4.0h respectively,then the optimal temperature was investigated by testing 24℃,28℃,32℃,37℃,42℃.The culture conditions were optimized,and bacteria were pelleted by centrifugation and disrupted by sonication,then the expression of fusion protein were assessed by SDS-PAGE,and identified using Western blotting.Results The expression level of GST-NS1 fusion protein was 28.5% by using the induce temperature at 37℃,the IPTG concentration of 0.3mmol/L for 5.0h induction.The expression amount of fusion protein was also 27.9%,when the IPTG concentration was 0.6mmol/L and the induce temperature was 37℃ for 4.0h induction and the expression level was 33.2%.The recombinant fusion protein GST-NS1 was expressed in a soluble form by SDS-PAGE.Analysis and the expression amount of soluble GST-NS1 was 25.1% using 0.6mmol/L IPTG concentration and 37℃ for 4.0h induction.Separated proteins on SDS-PAGE gel were transferred electrophoretically onto PVDF membrane,then the membrane incubated with anti-GST mouse monoclonal antibody,finally the expected band was shown.Conclusion Conditions for induction were optimized resulting in high expression of fusion protein GST-NS1 in soluble form.