利用温敏性凝胶的缓释作用促进变性溶菌酶复性

为了开发温敏凝胶在蛋白质复性方面的方法，利用N-异丙基丙烯酰胺温敏凝胶具有的缓释作用，对变性蛋白质进行了复性．考察不同合成条件下凝胶的性能，结果表明，在制胶温度为22℃、凝胶单体浓度为10％、交联度为5％制得的凝胶，具有较好的机械强度、较大的溶胀倍率、缩水倍率及良好的温敏性能．凝胶的缓释作用对变性还原溶茵酶辅助复性的影响表明，在复性酶终浓度为0．5mg／mL时，凝胶辅助蛋白质的复性收率可达68％，比直接稀释复性（收率为57％）高19％．特别是对高浓度蛋白质的复性（1．5mg／mL），凝胶辅助复性也获得了很好的效果，复性收率高出直接稀释复性37％，表现出温敏凝胶在实际蛋白质复性中的应用前景．     

血管抑素3A工程蛋白的柱复性与离子交换纯化

由大肠杆菌以包涵体形式表达的一种抗内皮细胞生长工程蛋白(A nti-ang iogen ic agent,简称3A)经变性,Sephacry l S-100 HR柱复性,SP Sepharose FF离子交换吸附纯化,SephadexG-25脱盐,获得复性率为53.47%,HPLC纯度为92.52%的3A活性蛋白。以猪髋动脉内皮细胞为受检细胞,表明纯化蛋白具有抑制内皮细胞生长的特性。     

Cause of Irreversible Inactivation of Aminoacylase in Urea Solution

ＩｎｔｒｏｄｕｃｔｉｏｎＡｍｉｎｏａｃｙｌａｓｅ (Ｎ ａｃｙｌａｍｉｎｏａｃｉｄａｍｉｄｏｈｙｄｒｏｌａｓｅ .ＥＣ 3.5.1.14 ) ,ｗｈｉｃｈｅｘｉｓｔｓｉｎｍａｍｍａｌｉａｎｋｉｄｎｅｙｓａｎｄｍｉｃｒｏｏｒｇａｎｉｓｍｓ ,ｃａｔａｌｙｚｅｓｔｈｅｒｅｖｅｒｓｉｂｌｅｈｙｄｒｏｌｙｓｉｓｏｆＬ ａｃｙｌａｍｉｎｏａｃｉｄｓ[1] .Ｔｈｅｅｎｚｙｍｅｉｓｄｉｍｅｒｉｃｗｉｔｈａｒｅｌａｔｉｖｅｍｏｌｅｃｕｌａｒｗａｓｓｏｆ86 0 0 0 ,ｃｏｎｔａｉｎｉｎｇｏｎｅＺｎ2 ｉｏｎｐｅｒｓｕｂｕｎｉｔｗｈｉｃｈｉｓ…     

纤维素内切酶Ⅲ再折叠过程中存在中间体

本文研究了尿素变性的纤维素内切酶Ⅲ的再折叠.通过更灵敏的方法—相图法,本研究发现在2 mol/L～4 mol/L尿素间,纤维素内切酶Ⅲ的再折叠过程中存在至少一个中间体,聚沉曲线也进一步验证了此中间体的存在.该中间体的存在个数可进一步通过蛋白动力学曲线得以确定.     

分子伴侣的研究进展

文章综述了分子伴侣特别是HSP70分子伴侣系统的结构、功能、作用机理及应用方面的研究进展。分子伴侣能结合和稳定另一种蛋白质的不稳定构象，促进新生多肽链的正确折叠，因而在辅助蛋白质复性以及免疫保护等方面有很重要的作用。HSP70分子伴侣能够帮助细胞内新生蛋白的折叠和跨膜运输、蛋白质多聚体结构的装配和解装配，并能在胁迫下维持蛋白质的特殊构象，防止未折叠的蛋白质变性和使聚集的蛋白质溶解复性。     

Chaperone-like features of bovine serum albumin: a comparison with α-crystallin

The chaperone behaviour of bovine serum albumin was compared with that of α-crystallin. The chaperone activity was assessed by measuring: (i) the ability to antagonize protein aggregation induced by heat; (ii) the capability to protect the activity of thermally stressed enzymes and (iii) the effectiveness in assisting the functional recovery of chemically denatured sorbitol dehydrogenase. Despite the lack of structural analogies, both proteins show several functional similarities in preventing inactivation of thermally stressed enzymes and in reactivating chemically denatured sorbitol dehydrogenase. As with α-crystallin, the chaperone action of bovine serum albumin appears to be ATP independent. Bovine serum albumin appears significantly less effective than α-crystallin only in preventing thermally induced protein aggregation. A possible relationship between chaperone function and structural organization is proposed. Together, our results indicate that bovine serum albumin acts as a molecular chaperone and that, for its particular distribution, can be included in the extracellular chaperone family. Received 29 August 2005; received after revision 23 September 2005; accepted 12 October 2005     

Alpha-crystallin: an ATP-independent complete molecular chaperone toward sorbitol dehydrogenase

-Crystallin, the major component of the vertebrate lens, is known to interact with proteins undergoing denaturation and to protect them from aggregation phenomena. Bovine lens sorbitol dehydrogenase (SDH) was previously shown to be completely protected by -crystallin from thermally induced aggregation and inactivation. Here we report that -crystallin, in the presence of the SDH pyridine cofactor NAD(H), can exert a remarkable chaperone action by favoring the recovery of the enzyme activity from chemically denaturated SDH up to 77%. Indeed, even in the absence of the cofactor, -crystallin present at a ratio with SDH of 20:1 (w:w) allows a recovery of 35% of the enzyme activity. The effect of ATP in enhancing -crystallin-promoted SDH renaturation appears to be both nonspecific and to not involve hydrolysis phenomena, thus confirming that the chaperone action of -crystallin is not dependent on ATP as energy donor.Received 28 October 2004; received after revision 22 December 2004; accepted 10 January 2005     

Expression，reflolding and preliminary characterization of recombinant snake venom metalloproteinases：Implication for the hemorrhagic mechanism

Two cDNAs encoding hemorrhagic snake venom metalloproteinase acutolysin A and non-hemorrhagic metalloproteinase(BR)were cloned into the expression vector pET-22b,respectively,and the corresponding two recombinant proteins,A-22b and BR-22b,were produced in inclusion bodies in E.coli BL21(DE3).The reombinant proteins were then subjected to solubilization,purification and refolding in vitro.A-22b showed hemorrhagic activity and fibronectin.Natural autolysin A had both hemorrhagic activity and proteolytic activity toward these substrates.BR-22b showed the proteolytic activities toward fibrinogen,but no hemorrhagic activity.In addition,two chimeric genes,C1 and C2,were constructed and cloned into pET-22b,and the corresponding recombinant proteins,C1-22b and C2-22b,were also expressed in inclusion bodies.C1-22b involved N-terminal 110 amino acidos of BR and C-terminal 95 amino acids of acutolysin A,while C2-22b contained N-terminal 108 amino acids of acutolysin A and C-terminal 112 amino acids of BR. The biological activities of A-22b and BR-22b,respectively.Our results suggested that N-terminal major subdomain of a snake venom metalloproteinase might play a key role in hemorrhagic activity and have an appreciable effect on the selectivity for protein substrates.     

降钙素受体药物靶点区域的表达、复性及鉴定

本实验截取了人降钙素受体N端胞外结构域涉及药物靶点的第20~160位氨基酸残基区域对应的编码序列,根据大肠杆菌偏爱密码子优化后,人工合成相应的基因,运用分子克隆技术将所合成的基因克隆到pET22b(+)载体,重组质粒转入大肠杆菌BL21(DE3)中原核表达.结果表明,Nt-CTR蛋白质以包涵体形式表达,所得包涵体蛋白质通过高效的复性方法,最终得到了大量纯化的可溶性Nt-CTR蛋白质.融合蛋白质通过Western-Blot鉴定表达正确.本研究为CTR的结构研究和CTR相关疾病的药物筛选提供了基础.