Construction And Preliminary Application Of The Prokaryotic Expression Vector For Soluble Expression Of Protein

Due to the advantages of producing recombinant protein in the E.coli, such as low cost, short period and being carcinogenic-gene-free, the E. coli is becoming the main tool used in gene engineering to produce cytokine and peptide medicine. While most heterogenous genes are likely to express proteins in the form of inclusion body in the E.coli, which leaves the refolding difficulty and the cost increased. The prokaryotic expression systems have no ability to modify productions of translation. Its intracellular surrounding is too reductive for the recombinant protein to form correct disulfide and quaternary structure, resulting in the low activity of the recombinant protein. So it is the key problem for the gene engineering to get the recombinant protein with high solubility and similar native activity in the E.coli. The Dsb proteins in the E.coli periplasm can make the cytoplasm high oxidation which will help the formation of disulfide of fusion protein. The overexpression of the Dsb proteins and the target protein can localize the target protein in cytoplasm and help it to form correct disulfide and tertiary structure.To obtain the recombinant protein in E.coli with high solibility, based upon the published result about Dsb family, we successfully constructed the coexpression vector, pET-SWGl-SWG2, which can express both human NGF (nerve growth factor β-subunit) and a cyclic 7 peptide-fused with human IgG Fc fragment in a soluble form. We first constructed the vector pET-SWGl-SWG2-NGF and pET-SWGl-SWG2-C7+Fc, and then optimized the expression condition, achieving the expression of these two recombinant protein in E.coli in soluble form. The restriction site of thrombin between the chaperone and the target protein permitted the separation and purification of these two target proteins. Biological assays confirmed that both of these two target proteins had similar native protein activities. Our result may be very useful in the prokaryotic expression of many other cytokines and peptides. It may also contribute to the research on the prokaryotic expression mechanism.