Study On Prepration And Function Of Recombinant Human Betaine-homocysteine S-methyltransferase(rhBHMT)

Objective: To establish one kind of biology engineering technology for preparing pharmaceutical proteins of human recombinant betaine-homocysteine methyltransferase (rhBHMT); to obtain highly purified rhBHMT with stable biological activity and then confirm its pharmacodynamic effect in preventing and treating hyperhomocysteinaemia.Methods: The gene of rhBHMT was cloned by Polymerase chain reaction technology and the engineering strain were built and screened. rhBHMT was expressed efficiently as soluble protein in E. coli BL21 and purified by DEAE Sepharose Fast Flow chromatography column and the original CM cation Chromatography. Its relative molecular weight, purity and concentration were analyzed by SDS-PAGE, HPLC, Lowry assay. rhBHMT activity in vitro was detected by sodium nitroprusside coloration method. Then, the animal model of hyperhomocysteinemia was created in wistar rats by using restraint stress and high-methionine diet intervention. After injecting with different does rhBHMT, the plasma HCY levels of hyperhomocysteinemia rats were observed to evaluate its biological functions.Results: The engineering strain was constructed successfully in which rhBHMT was expressed efficiently as soluble protein, and the purity of acquired rhBHMT protein reached more than 95%. The contention of acquired rhBHMT protein was from 0.01to1.0 mg / ml, its relative molecular mass was about 43kb, and could be stored stably in 20mmol / L PH7.0 sodium phosphate buffer solution under -20℃. One method for detection of rhBHMT activity in vitro was developed by sodium nitroprusside coloration with the superiority of more simple, reproducible, stable and high consistency with the results from amino acid determinator. The activity of rhBHMT protein in vitro was about 70 U / mg. When hyperhomocysteinemia rats developed by restraint stress and high-methionine diet were injected with rhBHMT, the plasma homocysteine level reduced significantly in dose-effect dependent manner.Conclusion: The engineering strain expressing rhBHMT was constructed successfully by biology engineering technology, and the methods for separation and purification of the rhBHMT protein were established. After obtained the highly purified rhBHMT, its physical and chemical property and activities were evaluated in vitro. In hyperhomocysteinemia rats, the recombinant rhBHMT showed its biological effect on reducing plasma homocysteine level. These studies provide a scientific basis for BHMT as a bioengineering drug to treat hyperhomocysteinemia.