Pyrococcus furiosus小分子热休克蛋白Pfu-sHSP的克隆达和纯化

用PCR扩增嗜高温菌Pyrococctu furiosus的小分子热休克蛋白基因后,克隆于质粒pT7473中.该小分子热休克蛋白Pfu-sHSP含有较多的稀有密码子,在大肠杆菌BL21(DE),几乎无表达,而在带有大肠杆菌稀有密码子AGA(arg)AGG(arg),AUA(ile)和CUA(leu)的BL21-Codonplus(DE)3-RIL中能大量表迭.大量表达Pfu-sHSP蛋白的细胞用超声波破碎后,离心沉淀用85℃水浴20 min,再离心,上清液再以Q Sepha-rose Fast Flow阴离子交换和S-200凝胶过滤层析进一步纯化,可以得到90%以上的蛋白.     

What’s new in the renin-angiotensin system?

Angiotensin-converting enzyme (ACE) is a zinc- and chloride-dependent metallopeptidase that plays a vital role in the metabolism of biologically active peptides. Until recently, much of the inhibitor design and mechanism of action of this ubiquitous enzyme was based on the structures of carboxypeptidase A and thermolysin. When compared to the recently solved structures of the testis isoform of ACE (tACE) and its Drosophila homologue (AnCE), carboxypeptidase A showed little structural homology outside of the active site, while thermolysin revealed significant but less marked overall similarity. The ellipsoid-shaped structure of tACE, which has a preponderance of -helices, is characterised by a core channel that has a constriction approximately 10 Å from its opening where the zinc-binding active site is located. Comparison of the native protein with the inhibitor-bound form (lisinopril-tACE) does not reveal any striking differences in the conformation of the inhibitor binding site, disfavouring an open and closed configuration. However, the inhibitor complex does provide insights into the network of hydrogen-bonding and ionic interactions in the active site as well as the mechanism of ACE substrate hydrolysis. The three-dimensional structure of ACE now paves the way for the rational design of a new generation of domain-selective ACE inhibitors.