THE EXPRESSION OF HUMAN MD-2/GST FUSION PROTEIN IN E. coli / 解放军医学杂志

ABSTRACT

To design and construct an expression vector,PGEX-4T-1/MD-2, and to express human myeloid differentiation protein-2(hMD-2) and glutathione-S-transferase (GST) fusion protein in E. coli, the EcoRI/SalI sites and stop code were incorporated into the hMD-2 encoding fragment by PCR. After digesting with EcoRI/SalI, the hMD-2 encoding fragment was cloned into the expression vector PGEX-4T-1 at the corresponding sites. The positive clones selected with PCR and restriction endonuclease digestion were sequenced and the expression of GST/hMD-2 fusion protein in E. coli BL21 was analyzed with SDS-PAGE after induced by 0.4mmol/L IPTG for 3 to 5 hours. The hMD-2 encoding fragment containing stop code was correctly inserted into the expression vector PGEX-4T-1 and this was confirmed by PCR, double-enzyme identification and sequencing. The SDS-PAGE analysis indicated that the GST/hMD-2 fusion protein was successfully expressed in E. coli and the yield of the fusion protein was 30 percent of bacterial total protein. The construction of the expression vector PGEX-4T-1/hMD-2 and the expression of fusion protein GST/hMD-2 in E. coli would be useful for further investigation of MD-2.