Yeast Adenosylmethionine Synthase Gene Cloning And Expression

S-adenosylmethionine (SAM) is a naturally occurring molecule, which widely distributes throughout tissues and fluids. It has been one of the very important pharmaceuticals because of its essential role in transmethylation, transsulphuration and transaminopropylation. SAM is currently used in medical and pharmacological areas due to its therapeutic potential in liver damage and affective disorders et al.The gene of sam2 encoded S-adenosylmethionine synthetase was amplified from S.cerevisiae NL365 by PCR and was cloned into vector pMD18-T to construct the recombinant plasmid pT-sam2; Then, it was subcloned into NcoI/EcoRI sites of pET20b(+) to construct the recombinant plasmid pETO-sam2. Plasmid pETI-sam2 was constructed from the products which were amplified by the whole plasmid PCR from pETO-sam2,digested with NdeI and self-ligated by T4 ligase.pETO-sam2 is lethal to E. coli BL21 (DE3), however, pETI-sam2 can be expressed successfully. After 6h induction by lmM IPTG and 0.8% L-methionine, the yield of SAM in crude extracts from recombinant strain BL21 (DE3) (pETI-sam2) was 1.273g/L, exhibited a 300-fold improvement over its parent. The expression products exists mainly in the form of inclusion body, and the relative molecular weight of which is proximately 43kD. And the quantity of the recombinant protein was about 16% of total bacterial protein.