A method for reducing protein polymers formed during denaturation and renaturation of inclusion bodies

Abstract

Objective To optimize protein preparation process and reduce polymers formed during denaturation and renaturation of inclusion bodies. Methods The inclusion bodies were harvested, denatured and renatured after bacteria cultivation and wall-broken according to original process. Then, solid ammonium sulfate was added directly to renaturation solution of inclusion bodies (optimized process). The precipitate was removed by centrifugation. Supernatant was collected for preparing crude protein product. The crude products prepared by the original and optimized processes were compared in terms of protein-binding capacity of chromatographic gel, protein recovery rate, purified protein amount and cytotoxic activity. Results By adding a step of 30% saturated ammonium sulfate precipitation, the optimized process increased protein-binding capacity of 1 ml chromatographic gel from 1 mg to 20 mg, the protein recovery rate of unit volume fermentation fluid from 10% to 90%, and the amount of protein purified out of 400 ml fermentation fluid from 3 mg to 15 mg. The mean 50% inhibitory concentration of crude products decreased from 0.077 99 μg/ml to 0.045 69 μg/ml. Conclusion Adding ammonium sulfate in renaturation solution can effectively reduce polymers. Key words: Recombinant fusion proteins; Polymers; Ammonium sulfate