Alvarez L, Mingorance J, Pajares M A, Mato J M
Instituto de Investigaciones Biomédicas, C.S.I.C., Madrid, Spain.
Biochem J. 1994 Jul 15;301 ( Pt 2)(Pt 2):557-61. doi: 10.1042/bj3010557.
A cDNA containing the complete coding sequence for rat liver S-adenosylmethionine synthetase was cloned into the prokaryotic expression vector pT7-7 and expressed in Escherichia coli BL21(DE3). A major additional band corresponding to a protein of 48 kDa was detected on SDS/PAGE after induction with isopropyl beta-D-thiogalactopyranoside. This protein was distributed in both the soluble and insoluble fractions and accounted for approx. 30% of the total bacterial protein. The soluble enzyme was fully active, as revealed by assays in vitro of S-adenosylmethionine synthetase activity. In addition, transformed bacteria exhibited highly increased levels of intracellular S-adenosylmethionine. Two active forms of the recombinant enzyme, with apparent molecular masses of 210 kDa and 110 kDa, were detected when cytosolic extracts of the transformed cells were fractionated by gel-filtration chromatography. It is concluded that the expressed S-adenosylmethionine synthetase polypeptide assemble as tetramers and dimers.
将含有大鼠肝脏S-腺苷甲硫氨酸合成酶完整编码序列的cDNA克隆到原核表达载体pT7-7中，并在大肠杆菌BL21(DE3)中表达。用异丙基β-D-硫代半乳糖苷诱导后，在SDS/PAGE上检测到一条对应于48 kDa蛋白质的主要额外条带。该蛋白质分布于可溶性和不溶性部分，约占细菌总蛋白的30%。体外测定S-腺苷甲硫氨酸合成酶活性表明，可溶性酶具有完全活性。此外，转化细菌的细胞内S-腺苷甲硫氨酸水平显著升高。当通过凝胶过滤色谱对转化细胞的胞质提取物进行分级分离时，检测到重组酶的两种活性形式，表观分子量分别为210 kDa和110 kDa。结论是，表达的S-腺苷甲硫氨酸合成酶多肽组装成四聚体和二聚体。
