大腹园蛛梨状腺丝重组蛋白的表达纯化与纺丝

圆网蜘蛛能分泌生产六种不同的蛛丝纤维和一种粘胶,其中梨状腺丝参与附着盘的形成,蜘蛛利用附着盘将蜘蛛丝固定于物体表面.迄今为止,相对于广泛研究的主壶腹腺丝,鲜有关于重组梨状腺丝蛋白与重组梨状腺丝力学性能的研究报道.本研究以大腹园蛛梨状腺丝蛋白(PySp1)一个重复单元的编码DNA为模板,通过无缝克隆技术构建能够表达含有2...     

大腹园蛛次壶腹腺丝的表达

基于大腹园蛛次壶腹腺丝Minor Ampullate Spidroin全长编码基因最新报道,研究了该基因的表达。利用PCR扩增该基因重复区一段长1 348 bp的片段P1,融合his-tag标签,构建酵母表达载体,在毕赤酵母菌GS115进行表达。同时构建大肠杆菌表达载体,在大肠杆菌BL21(DE3)中进行表达。SDS-PAGE和Western blotting检测结果表明,P1在两种表达系统中均可实现表达。研究结果显示:P1在GS115中的表达经优化后产量、产率有较大提高,且远高于BL21(DE3)中的表达,相应的纯化效率GS115也远高于对照BL21(DE3)的表达。研究表明酵母表达系统更适合重复度高、且富含Gly/Ala的天然蛛丝蛋白基因的表达,为表达全长天然MiSp编码序列提供前期实验基础,也为大规模蛛丝蛋白的重组表达建立了平台。     

大腹园蛛鞭毛样丝蛋白cDNA克隆

应用RT-PCR技术,从大腹圆蛛（Araneus ventricosus)壶腹腺中扩增出鞭毛样丝蛋白基因（flagelldid-form silk protein gene),经1.5%琼脂糖凝胶电泳分离,WizardPCR Preps DNA Purification System回收后,将其克隆在pGEM-T载体中,经限制性核酸内切酶鉴定和核苷酸序列分析证实,构建的重组擀粒pSF1中含有蜂蛛鞭毛样丝蛋白基因,且含有3个重复序列。     

蜘蛛大壶状腺丝蛋白基因的克隆和原核表达

以悦目金蛛(Argiope amoena)丝腺SMARTRACEcDNA文库为模板进行RT-PCR,克隆了1条大壶状腺丝蛋白(major ampullate spidroin,MaSp)基因cDNA序列。该条cDNA序列编码的氨基酸序列可区分为两部分(1)富含丙氨酸的片段和富含甘氨酸的片段相间排列构成的重复氨基酸序列区,并且富含甘氨酸的片段中有脯氨酸分布;(2)约100个氨基酸残基组成的C末端非重复氨基酸序列区。把MaSp基因cDNA序列亚克隆到质粒pET28b( )中,构建原核表达质粒pET28b( )-MaSp,表达质粒转化大肠杆菌BL21(DE3),用IPTG诱导表达。SDS-PAGE、氨基酸组成测定和N末端氨基酸序列测定的结果表明,表达产物为重组MaSp,表达量约为40mg/L。还对C末端非重复氨基酸序列对重组MaSp在水媒介中溶解性的影响进行了探讨。     

大腹圆蛛主壶腹腺cDNA文库构建和丝蛋白基因筛选

首次通过反转录-置换法和使用pUC18质粒成功构建大腹圆蛛（Araneus ventricosus)主壶腹腺（major ampullate gland)cDNA基因文库,并以鸟枪法从中筛选出具有典型重复结构的大腹圆蛛主壶腹丝蛋白cDNA基因AvF1,大小为1744bp,编码区为1572bp,编码氨基酸524个,分子量为42489.55Da,典型的重复结构为（GGP）nGGX。与现有已知的蛛丝蛋白基因中三带金蛛（Argiope trifas-ciata)鞭毛样丝基因（AtfF)有最高的同源性69.3%。大腹圆蛛主壶腹腺cDNA文库的构建和蛛丝蛋白新基因的克隆,为提供大腹圆蛛蛛丝蛋白基因背景和进一步研究蛛丝蛋白奠定了基础。     

大腹园蛛大壶状腺丝蛋白-1基因部分cDNA的克隆和序列分析

大腹园蛛大壶状腺表达拖丝蛋白新基因的克隆, 为进一步研究蛛丝蛋白基因以及人工表达蛛丝蛋白提供参考依据。文章利用“通用方法”即反转录—置换法构建大腹园蛛(Araneus ventricosus)大壶状腺(Major ampullate gland) cDNA文库, 并筛选出具有典型重复结构的大腹园蛛大壶状腺丝蛋白-1部分cDNA序列AvMaSp1 (GenBank登录号: AY177203)。该部分序列大小为1 408 bp, 编码区为1 288 bp, 编码氨基酸429个, 预测分子量为34.07 kDa, 典型的重复结构为 (GA)nAm(GA)N, 与十字园蛛(Araneus diadematus)丝蛋白基因ADF-1 (GenBank登录号: ADU47853)同源关系最近, 一致性为75.0%。     

大腹园蛛主壶腹腺蛛丝蛋白末端结构域的克隆表达

牵引丝(dragline silk)由主壶腹腺蛛丝蛋白(major ampullate spidroin, MaSp)组成,是蜘蛛丝中强度最好的丝,同时具有极佳的生物相容性和可降解性,因此引起研究者的研究热潮。目前关于大腹园蛛MaSp结构和成丝机理方面的研究甚少,限制了其仿生应用。本文以大腹园蛛牵引丝的组成蛋白质之一MaSp1为研究对象,通过锚定PCR的方法首次获取了大腹园蛛MaSp1 NT的完整编码基因,并对其进行了克隆、表达、纯化,产量可达60 mg/L;同时对该MaSp1的CT进行表达纯化,产量可达80 mg/L。另外,通过CD色谱分析了MaSp1 NT和CT的二级结构,结果表明二者的二级结构均以α-螺旋为主。上述结果为大腹园蛛MaSp1的结构和成丝机理研究奠定了基础。     

盐离子对次壶腹腺丝蛋白重组模块的影响

为研究不同生理环境对蛛丝蛋白组装及成丝的影响,首次以MiSp序列为对象,研究其NTR1SR2CT重组模块在不同种类(浓度)盐离子条件下的聚集和成纤维特性及其在成纤维过程中二级结构的变化。基于大腹园蛛MiSp全长序列构建NTR1SR2CT模块,并在大肠杆菌Escherichia coli BL21中成功表达。借助8 mol/L尿素裂解包涵体进行变性纯化得到NTR1SR2CT重组蛋白。NTR1SR2CT重组蛋白二级结构主要为无规则卷曲(Random coil)或α螺旋(Helix),在自然成丝及冻干过程中部分random coil或helix转变为β折叠(β-sheet),甲醇能促进该转变过程。另外,钾离子和磷酸根离子有利于NTR1SR2CT重组蛋白聚集从而促进丝纤维的形成。研究结果为成丝机理研究奠定了基础,同时也为工业化生产高品质的蛛丝纤维提供了条件。     

大腹园蛛丝腺的氨基酸组成分析

通过解剖大腹园蛛,获得５种丝腺体,并对各丝腺蛋白进行了氨基酸组成分析和蛋白质相对分子质量测定,比较了不同丝腺体蛋白质组成的差异,发现不同丝腺体蛋白质氨基酸组成中Ｇｌｙ和Ｇｌｕ均有较高含量,而Ｓｅｒ的含量并不高,壶状腺中含有较高的Ｐｒｏ,而管状腺中含有较高的Ａｒｇ．同时发现不同丝腺体蛋白质的相对分子质量存在较明显差异,例如集合腺中不同相对分子质量的蛋白质分布较均匀,而在葡萄状腺中大分子质量的蛋白质明显高于小分子质量蛋白质．     

人Fas N端融合蛋白的原核表达、纯化及初步分析

目的：对Fas蛋白N端的30～108位肽段进行原核表达,制备可溶性蛋白,并完成二级结构分析。方法：利用BLAST程序进行同源性分析,确定所要构建的FasN端半胱氨酸富集结构域（CRD）,PCR扩增目的基因片段,将其克隆入表达载体pGEX-6P-1,并转化大肠杆菌BL21（DE3）,于16℃用0.2mmol/L的IPTG诱导25h表达GST-Fas融合蛋白;用Glu-tathione Sepharose 4B柱分离GST-Fas融合蛋白,用PreScission蛋白酶切去GST标签,将洗脱的蛋白样品经Superdex 75凝胶预装柱进一步纯化;对所获得的可溶性、高纯度蛋白进行圆二色谱分析。结果与结论：获得重组质粒pGEX-6P-1-fas,并在大肠杆菌中可溶性表达了FasN端CRD功能结构域蛋白;经亲和层析、酶切、分子筛层析后,获得了可溶的、高纯度的FasN端CRD功能结构域蛋白;圆二色谱结果揭示目标蛋白富含β-折叠,为进一步研究Fas的结构和功能奠定了基础。     

mLST8在大肠杆菌中的表达、纯化及鉴定

目的:表达并纯化mLST8蛋白。方法:PCR扩增mLST8的编码cDNA,克隆到pET-28a(+)表达载体,将重组质粒pET-28a-mLST8转化大肠杆菌BL21(DE3)感受态细胞,在IPTG诱导下表达目的蛋白;提取包涵体,用Ni2+亲和层析纯化目的蛋白,稀释和透析相结合进行复性,对复性蛋白进行阴离子交换层析、分子筛层析,将纯的复性mLST8进行肽指纹质谱鉴定和圆二色谱分析。结果:酶切和DNA测序证明pET-28a-mLST8表达质粒构建无误,并在大肠杆菌中得到高效表达;通过Ni2+亲和层析、复性、离子交换层析和分子筛层析获得了较高纯度的复性蛋白,肽指纹质谱鉴定为mLST8;mLST8蛋白的二级结构[α螺旋为18.2%,β折叠为52.3%(其中平行结构为12.1%,反向平行结构为40.2%),β转角为20.7%,无规则卷曲为39.9%]表明其为典型的β折叠结构。结论:在大肠杆菌中表达了重组mLST8蛋白,复性获得了二级结构准确的mLST8,为进一步研究mLST8的晶体结构与功能奠定了基础。     

大腹圆蛛Fosmid文库的构建及MaSp基因筛选方法的探讨

介绍了构建大腹圆蛛Fosmid基因组文库及牵引丝蛋白(MaSp)基因克隆筛选的全过程.采用改良CTAB法提取大片段基因组DNA,通过自主构建的电洗脱核酸回收装置分离回收30～40kbDNA片段,经补平磷酸化、与pCC2FOS载体连接、体外包装和转染EPI300TM-T1R,首次构建了无偏向性的大腹圆蛛Fosmid文库,其滴度为4.5×105cfu/mL,覆盖基因组倍数为10.以α-32P标记寡核苷酸探针对文库进行初步筛选,获得含MaSp基因的12个阳性克隆.该文库符合Fosmid文库的品质要求,为进一步筛选并研究大腹圆蛛MaSp基因序列奠定了基础.     

大腹园蛛鞭状腺蛛丝蛋白N端结构域的克隆表达及圆二色谱分析

鞭状腺蛛丝是6种蛛丝中延展性最好的丝,由鞭状腺蛛丝蛋白(FlSp)构成,具有极大的潜在应用价值。目前,有关FlSp的末端功能模块(NT和CT)编码基因的报道极少,且其结构和功能均尚未明确,这在一定程度上限制了鞭状腺蛛丝的仿生。现利用5′-RACE的方法,以大腹园蛛总RNA和基因组DNA为模板,首次获取了大腹园蛛FlSp NT模块(FlSp_A.v-NT)的完整编码基因,并对其成功进行了克隆、表达及纯化,产量可达60 mg/L;同时,利用圆二色谱(circular dichroism,CD)对FlSp_A.v-NT的二级结构进行了分析,结果显示其主要以α螺旋构象存在,这是首次对FlSp NT的二级结构进行探索,为后续FlSp NT结构功能的研究提供了材料,奠定了研究基础。     

Preparation of recombinant human thymic stromal lymphopoietin protein

Human thymic stromal lymphopoietin (hTSLP) protein plays a central role in inflammation. Characterizing properties of hTSLP requires a recombinant overexpression system that produces correctly folded, active hTSLP. In this report, an efficient overexpression system for the production of hTSLP was developed. We constructed expression plasmids of the full-length hTslp gene with or without the signal peptide and transformed the plasmids into Escherichia coli. The design of the recombinant proteins included an N-terminal His-tag, which facilitated purification. An affinity gradient elution method was used to improve recovery and concentration levels of denatured hTSLP, with 90% purity observed following affinity chromatography. Refolding of the denatured hTSLP was tested using four different protein refolding approaches. The optimal refolding conditions involved stepwise buffer exchanges to reduce the urea concentration from 4 to 0?M in 50?mM Tris (pH 8.0), 1?mM EDTA, 50?mM NaCl, 10% glycerol, 400?mM L-Arg, 0.2?mM oxidized glutathione, and 2?mM reduced glutathione. The activity of the refolded recombinant hTSLP protein was measured by an ELISA assay. Interestingly, the presence of N-terminal signal peptide inhibited the overexpression of hTSLP in E. coli. The amount of recombinant hTSLP protein purified reached a level of 2.52?×?10^?3?mg/L.     

Spectroscopic investigation of the structure of protoxin protein isolated from Bacillus thuringiensis contacted with minerals

Effects of minerals on the conformational changes of protoxin isolated from Bacillus thuringiensis were investigated by circular dichroism and fluorescence spectroscopy. Contact of the protoxin with attapulgite, montmorillonite and kaolinite for 3 h resulted in no significant changes in the spectra of circular dichroism and a slight decrease in the fluorescence intensity. There were significant changes in spectra of circular dichroism of protoxin after desorption in comparison to the native protoxin. The fluorescence intensity of protoxins desorbed from minerals retained 77.5, 63.7 and 60.4% of intensity of native protoxin, respectively. The influential extent of desorption on the secondary structure was higher than that of contact.     

拟蜘蛛拖丝蛋白基因人工合成及其原核表达

根据GenBank上蜘蛛拖丝蛋白基因序列(AY555585和AH015065)、拖丝蛋白基因的结构特点和密码子的简并性,设计378 bp的拖丝蛋白基因单体,并对其人工聚合成二聚体.Primer5.0分析结果表明,决定拖丝弹性和抗拉力的氨基酸(Gla和Ala)含量(41.43和18.22)接近天然丝蛋白氨基酸含量(42.81和26.32);利用Antheprot软件对二聚体编码氨基酸的二级结构预测结果显示,与丝蛋白的氨基酸链相近,由2个相同的部分排列而成,即β-片层(占48%)、6个α-螺旋间隔(占15%)、散在的转角(占12%)和若干不规则卷曲(占25%).将二聚体与pET-28a( + )连接构建原核表达载体,IPTG诱导重组菌体裂解物经SDS-PAGE电泳可检测到相对分子质量为26.6×103 kD的重组蛋白.上述结果为进一步开展有关转蜘蛛拖丝蛋白基因在绵羊被毛中表达的研究奠定了基础.     

Use of [Cit312,314] filaggrin (306–324) analogue for the diagnosis of rheumatoid arthritis. Conformational study by circular dichroism and fourier transformed infrared spectroscopy

Summary Antifilaggrin autoantibodies (AFA) have described to be the most specific markers of rheumatoid arthritis (RA) and epitopes containing citrulline within the sequence of filaggrin identified as major antigenic sites recognised by AFA. In this paper we confirm that citrulline is an essential constituent of filaggrin related antigenic determinants recognised by RA specific autoantibodies, thus having an important role for the development of filaggrin peptides based serological tests. Moreover, we describe our findings on the comparative conformational analysis of filaggrin (306–324) peptide sequence and an analogue in which two arginines were simultaneously substituted by citrulline carried out by Circular Dichroism (CD) and Fourier-Transform Infrared Spectroscopy (FTIR). Results might contribute to the understanding of the structural features that may be important to explain the enhanced binding characteristics of citrullinated peptides to the autoantibodies in rheumatoid arthritis.     

Extreme heat- and pressure-resistant 7-kDa protein P2 from the archaeon Sulfolobus solfataricus is dramatically destabilized by a single-point amino acid substitution

This study reports the characterization of the recombinant 7-kDa protein P2 from Sulfolobus solfataricus and the mutants F31A and F31Y with respect to temperature and pressure stability. As observed in the NMR, FTIR, and CD spectra, wild-type protein and mutants showed substantially similar structures under ambient conditions. However, midpoint transition temperatures of the denaturation process were 361, 334, and 347 K for wild type, F31A, and F31Y mutants, respectively: thus, alanine substitution of phenylalanine destabilized the protein by as much as 27 K. Midpoint transition pressures for wild type and F31Y mutant could not be accurately determined because they lay either beyond (wild type) or close to (F31Y) 14 kbar, a pressure at which water undergoes a phase transition. However, a midpoint transition pressure of 4 kbar could be determined for the F31A mutant, implying a shift in transition of at least 10 kbar. The pressure-induced denaturation was fully reversible; in contrast, thermal denaturation of wild type and mutants was only partially reversible. To our knowledge, both the pressure resistance of protein P2 and the dramatic pressure and temperature destabilization of the F31A mutant are unprecedented. These properties may be largely accounted for by the role of an aromatic cluster where Phe31 is found at the core, because interactions among aromatics are believed to be almost pressure insensitive; furthermore, the alanine substitution of phenylalanine should create a cavity with increased compressibility and flexibility, which also involves an impaired pressure and temperature resistance. Proteins 29:381–390, 1997. © 1997 Wiley-Liss, Inc.     

The osmolyte betaine promotes protein misfolding and disruption of protein aggregates

In this work the effect of betaine on the structure and aggregation of the GST-GFP fluorescent fusion protein was studied by different complementary techniques, including electron microscopy, dynamic light scattering, circular dichroism, and FTIR spectroscopy. Although osmolytes are known to be protein stabilizers in vivo, the effect of betaine on the structure and aggregation of our model protein was found to be strictly concentration dependent. We demonstrated that, by changing betaine concentration, it was possible to tune the formation of protein soluble assemblies and insoluble aggregates, as well as to disaggregate preformed aggregates. In particular, at a critical concentration of betaine between 5 and 7.5 mM, the protein precipitated into macroscopic prefibrillar structures, rich in intermolecular beta-sheets, which were found to bind thioflavine T and to be inaccessible to protease. Instead, at higher betaine concentration (10-20 mM) the misfolded protein lost its fluorescence, but formed soluble assemblies with hydrodynamic radius of about 16 nm. These structures displayed a reduced propensity to further aggregate under thermal treatment. In addition, betaine at this high concentration was also found to disrupt large preformed aggregates, obtained under different conditions, into protein soluble assemblies. It is the first time that a disaggregation process has been described for a chemical chaperone. A mechanism for the betaine concentration-dependent effect on protein misfolding, aggregation, and disaggregation is proposed and its possible physiological implications are discussed.