Human Secreted Phosphoprotein 2 (spp2) Cloning, Prokaryotic Expression And Activity Assay

Object:Secreted Phosphoprotein 2(SPP2) is a non-collagenous extracellular matrix protein first separated and purified from bovine cortical bone. As there is a phosphorylated serine-rich sequence between residues 131 and 139 of SPP2, which have a molecular mass of about 24kDa, the protein is also termed secreted phosphoprotein 24(SPP24). Previous studies suggested that one biological role of SPP2 might be to inhibit the cysteine protease. SPP2 also involved in bone formation and small amounts SPP2 were found in the fetuin-mineral complex (FMC) and may inhibit calcification, however its mechanism of action is still unknown. Our research object is to clone the DNA fragment of human mature protein of secreted phosphoprotein 2 into prokaryotic expression system by the technique of gene engineering and induce the expression of the recombinant protein, then obtain the recombinant protein with high purity and analyze its biological activity.Methods:Total RNA was extracted from human liver cancer tissue.. The target gene encoding the mature protein of human SPP2 was obtained by RT-PCR, then cloned into the prokaryotic expression vector pET-22b(+). The recombinant plasmid pET-22b(+)-SPP2 was transformed into E.coli BL21(DE3) where it was induced to express protein by IPTG The recombinant protein was purified by Ni-NTA column chromatography and detected and confirmed by SDS-PAGE as well as Western-blot. Its biological activity was evaluated by inhibiting the protease activity of papain. The recombinant protein was heated at different temperature for different time to analyze the effect of heat on its inhibition of papain.Results:Sequence and restriction analysis reveal that the target gene encoding the mature protein of human SPP2 was cloned into pET-22b(+). After induction by IPTG, there was a band of protein with a molecular weight of 25kDa detected on SDS-PAGE and soluble expression was acquired by optimizing the conditions of expression. The purity of the target protein was beyond 90% and its His tag antigen activity was examined by Western-blot. The recombinant protein play a role of inhibiting the protease activity of papain (casein as substrate). Its weight ratio was about 1:3.1 and its inhibition specific activity reached 2511U/mg. When the recombinant protein was heated, its inhibition activity decreased obviously with increasing temperature and time. Conclusion:In this study, the recombinant mature protein of human secreted phosphoprotein 2 with biological activity was obtained through gene engineering and E.coli expression system. Papain is a typical member of cysteine protease, we determined the inhibition of papain by recommbiant SPP2 and verified the presumption that SPP2 is an inhibitor of cysteine protease. All these results have facilitated further studies on the biological function and mechanism of SPP2.