Prokaryotic Expression, Purification And Study On Co-Renaturation Of Truncated Recombinant Protein HLA-G1

HLA(human leukocyte antigen)-G is a non-classical MHC(major histocompatibility complex) class I molecule involved in immunotolerance. It is selectively expressed on cytotrophoblasts at the maternal-feto interface, where it plays an important role in maternal tolerance of the semiallogenetic fetus. Recently, some reports showed that malignant melanoma cells exhibit a high level of HLA-G transcription and protein expression. Thus, they proposed that HLA-G may play a functional role in protecting tumor cells from NK destruction. This remains a highly controversial area. In contrast to classical HLA-Ia genes, HLA-G has limited polymorphism, as other HLA-Ib genes. Today, sixteen alleles have been described that give rise to only four productive proteins for HLA-G. The primary transcript of the HLA-G gene generates at least seven isoforms: four membrane-bound proteins (HLA-G1,-G2,-G3,-G4) and three soluble proteins (HLA-G5,-G6,-G7).On the basis of a recombinant cloning plasmids pGEM-T-HLA-G1, we constructed prokaryotic expression plasmids pET28a-sHLA-G1, which can express truncated HLA-G1 in E. coli. His-sHLA-G1 was purified for the further research.Humanβ2-microglobulin (hβ2m) is the light chain of major histocompatibility complex (MHC) class-Ⅰmolecule. Expression and purification of this protein in Escherichia coli (E. coli) is a prerequisite to the preparation of MHC-I molecule. We were presented the expression vector pET23a-hβ2m,in which the complete sequence ofβ2 m gene was inserted. Constant-yield expression of hβ2m was achieved in E.coli transformed with the expression vector ,and most of the recombinant hβ2m existed in the inclusion body after IPTG induction. The inclusion body was washed extensively and hβ2m in the inclusion body was solublized with 6 M urea. The hβ2m was refolded by dialysis and purified by ion-exchange chromatography (Q-Sepharose). Western blotting assay indicated that the polyclonal antibody against human nativeβ2m could react specifically with the recombinant protein. The purified protein appeared as a single band on both SDS-PAGE and Western blotting,indicating that it was chemical and antigenic pure.In the optimized expressing condition both strains, DE3-pET28a-sHLA-G and...