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How can soluble protein expression be improved?
When recombinant proteins are expressed at high levels in E. coli, they easily form insoluble inclusion bodies within the cells, causing trouble for subsequent purification and refolding. Moreover, the refolded protein often has low or no activity. Therefore, improving the soluble expression of recombinant proteins in E. coli is very necessary.
Optimization can be done from the following aspects:
1.Selecting an appropriate host cell.
2.Using suitable fusion tags.
3.Choosing an appropriate plasmid vector.
4.Optimizing culture and induction conditions.
What causes proteins expressed in the bacterial cytoplasm to form inclusion bodies?
Intracellular expression is the primary form of recombinant protein expression in E. coli. However, the E. coli cytoplasm has a reducing environment, which is unfavorable for the formation and stability of protein disulfide bonds. This leads to incorrect protein folding and the formation of insoluble inclusion bodies. Protein kinetic model studies indicate that the yield of active protein also depends on the rates of protein synthesis, folding, and aggregation. When foreign proteins are highly expressed in E. coli, if the aggregation rate of nascent polypeptide chains exceeds their folding rate, it leads to inclusion body formation.