Sharma A, O'Connell B C, Tabak L A, Bedi G S
Department of Oral Biology, State University of New York at Buffalo, USA.
Arch Oral Biol. 1995 Jul;40(7):639-44. doi: 10.1016/0003-9969(95)00016-i.
Cystatin S is a cysteine proteinase inhibitor that is transiently expressed during rat submandibular gland development and can be induced by isoproterenol in the adult. A cDNA for rat cystatin S which included the entire coding sequence of the secreted cystatin was cloned. A coding region of the cystatin gene was amplified by polymerase chain reaction and cloned into the pGEX-2T expression vector. The chimeric plasmid was transformed into Escherichia coli, and protein expression was induced by isopropyl-beta-D-thiogalactopyranoside. The expressed protein was purified from insoluble inclusion bodies after solubilization with urea and fast protein liquid chromatography on a MonoQ column. The purified recombinant cystatin reacted with antibodies to cystatin S purified from rat submandibular glands and showed an amino-terminal amino acid sequence identical to that of rat cystatin S. The recombinant protein exhibited papain inhibition activity comparable to natural cystatin. This was a successful expression and purification of a functionally and immunologically reactive recombinant cystatin from E. coli, an approach which will be used later towards generating recombinant variants to study the binding and functional domains of this cysteine protease inhibitor.
胱抑素S是一种半胱氨酸蛋白酶抑制剂，在大鼠颌下腺发育过程中短暂表达，在成年大鼠中可被异丙肾上腺素诱导表达。克隆了大鼠胱抑素S的cDNA，其包含分泌型胱抑素的完整编码序列。通过聚合酶链反应扩增胱抑素基因的编码区，并将其克隆到pGEX-2T表达载体中。将嵌合质粒转化到大肠杆菌中，用异丙基-β-D-硫代半乳糖苷诱导蛋白表达。用尿素溶解后，通过MonoQ柱快速蛋白质液相色谱法从不溶性包涵体中纯化表达的蛋白。纯化的重组胱抑素与从大鼠颌下腺纯化的胱抑素S抗体发生反应，其氨基末端氨基酸序列与大鼠胱抑素S相同。重组蛋白表现出与天然胱抑素相当的木瓜蛋白酶抑制活性。这是从大肠杆菌中成功表达和纯化出具有功能和免疫活性的重组胱抑素，该方法随后将用于产生重组变体以研究这种半胱氨酸蛋白酶抑制剂的结合和功能结构域。
