AiiA融合蛋白包涵体变性和复性的研究

通过IPTG诱导含有pGEXaiiA-B15质粒的工程菌使其大量表达AiiA融合蛋白.由于所表达的融合蛋白多为不可溶的包涵体,须经分离、变性溶解,再经过一个合适的复性过程才能实现变性蛋白的正确折叠,得到具有生物活性的蛋白.通过含N-十二烷基肌氨酸钠的缓冲液A溶液及尿素等变性剂使包涵体溶解,磷酸盐缓冲液透析复性.SDS-PAGE电泳表明包涵体已由不可溶转化成可溶的蛋白.抑菌试验证明复性后的AiiA蛋白对胡萝卜欧文氏软腐病菌引起的马铃薯软腐病有较明显的抑制作用.     

离子交换色谱流动相组成对核糖核酸酶的折叠影响

用离子交换色谱法（IEC）研究了还原变性核糖核酸酶（RNase A）折叠过程中，复性缓冲液种类及pH值、流动相中脲浓度及盐种类对还原变性核糖核酸酶复性的影响。发现pH为弱碱性，用PBS作为缓冲液能提高复性效率；当流动相中含有2．0mol／L脲时复性产率较高；洗脱剂用NaCl时活性回收率较高。     

重组基质金属蛋白酶MT5-MMP的表达、 纯化和复性

对已经构建成功并转化到大肠杆菌E.coli BL21(DE3)的基质金属蛋白酶MT5 MMP的催化结构域进行诱导表达, 得到分子量为21000, 包涵体形式的重组蛋白. 通过离子交换树脂对目的蛋白进行纯化后, 用凝胶过滤树脂对纯化后的蛋白进行柱上复性, 得到了具有较高活性的重组蛋白.     

人P53的原核表达及包涵体复性研究

通过检测血清中p53蛋白及其抗体变化诊断肿瘤的ELISA试剂盒的研发需要大量制备p53蛋白.本研究通过构建人野生型p53基因的原核表达质粒pET-32a-p53,在大肠杆菌中诱导表达得到p53包涵体蛋白,经包涵体变性、亲和纯化、复性得到可溶p53蛋白.发现利用8M尿素对包涵体进行变性溶解,经镍柱亲和纯化得到纯度超过95%的变性蛋白.尝试透析复性、稀释复性和柱上复性3种方法分别对p53变性蛋白进行复性.结果表明透析复性的复性率最高,这为原核表达制备p53提供了一种参考.本研究为ELISA法检测血清中p53蛋白及其抗体诊断肿瘤相关试剂盒研发奠定了基础.     

Increase Renaturation Yield of Reteplase Using the Recombinant Human Protein Disulfide Isomerase

Reteplase, the recombinant type of novel tissue plasminogen activator (t-PA) variant, is a promising thrombolytics in clinics. Expressed in the form of an inclusion body, reteplase consists of about 40% of the total intracellular proteins of Escherichia coli. The recombinant human protein disulfide isomerase (rhPDI) is used to increase the chance for the correct matching of the 18 hydrosulfide groups of the reteplase molecule in the renaturation process and it increase is the reteplase renaturation yield from 1%-2% to 15% - 20% with a the purity about 99% and the specific activity of 5(105 IU/mg is reached.This novel method can reduce significantly the cost of production.     

大肠杆菌表达的成骨蛋白-1的复性研究

带有rhOP-1／pBV221-的E．coli表达得到的rhOP-1以不溶的包涵体形式存在，用高浓度的变性剂溶解后，经过DEAE-FF纯化，得到高纯度的目的蛋白。利用各种不同方法对蛋白质进行复性，并对复性结果进行比较，发现添加氧化还原剂最有助于二聚体的形成，这是由蛋白质分子的结构和理化性质决定的。     

血管生长素衍生物His13Gly的基因工程研究

在人工合成的血管生长素基因的基础上，利用化学合成的寡聚核苷酸片段，采用掺Ｕ法对ＡＮＧ知性定位眯诱变，同位素杂交筛选到ＡＮＧ基因的衍生物ＩＳ１３Ｇｌ６突变子。     

铜绿微囊藻气囊结构蛋白GvpC的表达纯化与晶体生长研究

对GvpC进行了初步晶体学研究。首先对GvpC进行分子克隆,利用原核表达系统在体外进行异源表达;结合变复性和凝胶过滤层析的方法纯化GvpC;通过圆二色谱的方法鉴定GvpC的复性效果;运用坐滴蒸汽扩散法进行晶体初筛。结果表明：GvpC在大肠杆菌表达系统中表达为包涵体;通过变复性和凝胶过滤层析能纯化出纯度较高的蛋白;圆二色谱证实复性后的GvpC形成了正确的二级结构,复性效果较佳;通过晶体初筛获得了GvpC的晶体,后续即可通过解析GvpC的晶体结构而获得其三维结构。     

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     

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