Cloning, Expression And Affinity Purification Of Recombinant His Tag Proteins

In order to obtain over-expressed recombinant His Tag protein for high performance refolding strategy, polymerase reaction (PCR) was used for amplifying the gene encoding MDH from chromosome of E.coli DH5αand EGFP gene from vector pEGFP-C1 as well as DNA of RNase A from bovine pancreas. Then the PCR fragment was cloned into the expression vector pET28a to get recombinant plasmid (pET28a-MDH pET28a-EGFP and pET28a-RNase A). The positive recombinant, verified by restriction endonuclease digestion and sequence analysis, was transformed into host E.coli BL21(DE3). The engineering strains (aMN3 and aEN5) expressing proteins (MDH and EGFP) were over-expressed so as to find the optimized induced condition. aMN3 was growing in 4h at 37℃then induced 9h at 37℃with 1mmol/L IPTG. aEN5 was growing in 4h at 37℃then induced 3h at 37℃with 1mmol/L IPTG. Assayed by SDS-PAGE, foreign protein MDH was over-expressed with a yield of 63.0% and EGFP with a yield of 49.7%, mainly as IBs as well as an amount of soluble protein. The production can reach 153.3mg/(L culture) and 140.3mg/(L culture).As expression products were containing six His Tag, IMAC (Chelating SepharoseTM Fast Flow) was used for affinity separation and refolding MDH and EGFP. The result showed that IMAC can adsorb soluble recombinant protein and unfolding IBs dissolved by 8mol/L Urea. The recombinant protein can be well purified by eluting.