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How to select an appropriate host cell?
The main reason recombinant proteins often fail to fold correctly in the E. coli cytoplasm is the reducing environment, which disfavors disulfide bond formation. To address this, researchers have mutated genes of key enzymes in the main reducing pathways for intracellular expression. This has led to the construction of competent cells that favor disulfide bond formation and correct protein folding, such as Origami(DE3), Origami B(DE3), Rosetta-gami(DE3), Shuffle, etc.
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.