巨大芽孢杆菌分子伴侣GroES和GroEL新基因的克隆及同源性分析

巨大芽孢杆菌作为革兰氏阳性细菌的一种,是良好的重组蛋白的表达宿主.本研究利用PCR技术从巨大芽孢杆菌基因组克隆出一条1.9 Kb的基因片段.核酸序列分析结果表明,该片段全长1 984 bp,包含2个ORF,分别与芽孢杆菌来源的GroES和GroEL基因有高度的相似性.氨基酸序列比对发现,GroES蛋白与枯草芽孢杆菌来源的GroES蛋白氨基酸序列同源性为91%,GroEL蛋白氨基酸序列同源性为90%.     

GroEL的耗能分子伴侣机制

双环结构Gro EL及其辅分子伴侣Gro ES是目前研究得最深入的分子伴侣.然而,Gro EL/Gro ES帮助蛋白质折叠的一些关键理化机制,尤其是水解ATP,Gro EL发生构象改变,能否主动调节蛋白质错误折叠中间体的构象,以促进错误折叠中间体的复性,仍然存在争议.结合本研究组近年的工作,作者着力介绍Gro EL促进蛋白质折叠的主动解折叠机制.     

巨大芽孢杆菌淀粉酶基因的克隆及其在枯草杆菌中的表达

以λ噬菌体为载体,采用鸟枪法由B.megaterium基因组克隆得到了1个淀粉酶基因,并已被亚克隆到E.coli和B.subtilis中,其表达水平较B.megaterium高250倍。克隆株产生的淀粉酶对直链淀粉的早期水解产物主要为麦芽三糖和麦芽糖,随着水解时间的延长,又将它们转变为葡萄糖。同时能以麦芽三糖为底物水解为麦芽糖和葡萄糖。受体菌的平行提取物无上述水解活性。因而该酶被确定为糖化型α-淀粉酶。SDS-凝胶电泳法确定酶分子量为58000道尔顿。     

幽门螺杆菌分子伴侣GroEL相互作用蛋白分析

【目的】获得幽门螺杆菌(Helicobacter pylori,HP) GroEL结合蛋白质组构成谱,为进一步探究GroEL及其与相互作用蛋白在HP致病机制中的作用提供新思路。【方法】在构建HP GroEL原核表达重组大肠杆菌(Escherichia coli) BL21(DE3)⁽pET-28a(+)-groEL)基础上,纯化带有His标签的GroEL蛋白,与HP全菌蛋白提取液共孵育后,利用Protein G磁珠和抗His标签抗体免疫沉淀法对复合物进行捕获,然后对复合物中GroEL及其结合的蛋白质进行质谱法鉴定,根据主要功能对其进行分类,并完成蛋白质相互关系网络分析。【结果】对GroEL蛋白捕获成分进行分析,共鉴定出59种可能与GroEL结合的蛋白质,其中包括19种代谢酶类(KatA、GltA和AhpC等参与氧化还原相关酶类7种,PepA、RocF和HtrA等肽酶5种,以及2种参与脂肪代谢酶、2种参与ATP合成酶、2种尿素酶和HP17＿08079蛋白等)、15种外膜蛋白(黏附素BabA、SabA、HapA及其他膜蛋白等)、8种转录翻译相关蛋白(Tuf、RpoBC...     

巨大芽孢杆菌b-淀粉酶基因的克隆、表达和酶学性质分析

从巨大芽孢杆菌(Bacillus megaterium)的全基因组DNA文库中筛选出一个b-淀粉酶基因amyG, 分析测定了其核苷酸序列并进行了诱导表达; 其中amyG编码的蛋白有545个氨基酸、分子量为60.194 kD, 与已报道的巨大芽孢杆菌DSM319的b-淀粉酶序列有着94.5%的同源性。经氨基酸序列比较分析发现, AmyG从N末端到C末端依次由信号肽域、糖基水解酶催化功能域和淀粉结合域3个功能域组成。其中催化功能域里含有第14家族糖基水解酶常见的几个高度保守的酶催化活性区。经多步纯化, 重组酶的比活共提高了7.4倍, 获得凝胶电泳均一的蛋白样品; 经SDS-PAGE电泳测定, 酶AmyG的分子量为57 kD。该酶的最适反应温度为60oC, 最适反应pH为7.0; 在温度不超过60oC时, 酶活较稳定; AmyG能迅速降解淀粉生成麦芽糖, 属于外切b-糖苷酶。     

巨大芽孢杆菌β-淀粉酶基因的克隆、表达和酶学性质分析

从巨大芽孢杆(Bacillus megaterium)的全基因组DNA文库中筛选出一个β-淀粉酶基因amyG,分析测定了其核苷酸序列并进行了诱导表达;其中amyG编码的蛋白有545个氨基酸、分子量为60.194 kD,与已报道的巨大芽孢杆菌DSM319的β-淀粉酶序列有着94.5%的同源性.经氨基酸序列比较分析发现,AmyG从N末端到C末端依次由信号肽域、糖基水解酶催化功能域和淀粉结合域3个功能域组成.其中催化功能域里含有第14家族糖基水解酶常见的几个高度保守的酶催化活性区.经多步纯化,重组酶的比活共提高了7.4倍,获得凝胶电泳均一的蛋白样品;经SDS-PAGE电泳测定,酶AmyG的分子量为57 kD.该酶的最适反应温度为60℃,最适反应pH为7.0;在温度不超过60℃时,酶活较稳定:AmyG能迅速降解淀粉生成麦芽糖,属于外切β-糖苷酶.     

地衣芽孢杆菌2709碱性蛋白酶基因的克隆与序列分析

以地衣芽孢杆菌２７０９染色体ＤＮＡ为模板,应用聚合酶链反应方法扩增了碱性蛋白酶基因,ＰＣＲ反应产物在１％琼脂糖凝胶上表现为１条１．１ｋｂ的条带。该片段经电泳纯化后被克隆于大肠杆菌质粒ｐＢｌｕｅｓｃｒｉｐｔ ＳＫ上,获得ａｐｒＬ＾＋克隆株。ＤＮＡ序列分析表明,它与枯草杆菌蛋白酶Ｃａｒｌｓｂｅｒｇ具有很高的同源性,同样由编码信号肽、前导肽及成熟蛋白的３个部分构成。     

巨大芽孢杆菌α-淀粉酶基因核苷酸序列分析

巨大芽孢杆菌(Bacillus megaterium)AS1.127的淀粉酶基因的全碱基序列已被测定。结构基因由1982bp的单一开读框架组成。由DNA序列推测出的前体酶蛋白由659个氨基酸组成,N-端33个氨基酸为信号肽。成熟酶分子由626个氨基酸组成,分子量为68.676kD。该淀粉酶属糖化型α-淀粉酶。并与枯草杆菌(B.subtilis)168产生的糖化型α-淀粉酶之间有83.3%的同源性。分析发现两种菌产生的酶分子的N-端3/4的同源性为90.4%,而C-端1/4的同源性只有70%。序列排比结果说明在淀粉酶基因的趋异进化过程中,基因突变和遗传重组都曾起过作用。     

巨大芽孢杆菌青霉素G酰化酶基因的克隆和表达

我们分离到了一株产生分泌型青霉素G酰化酶的巨大芽孢杆菌(Bacillus megateriumBM1)。用pBR322作载体,将该菌的青霉索G酰化酶基因克隆到大肠杆菌(Escherichia coliMcl061)中,得到含有9．9kb插人片段的重组质粒pBmPA4。分析了该质粒的限制酶酶切图谱,并经体外缺失获得含4．9kb插入片段的质粒pBmPA5。pBmPA4和pBmPA5在E·coliMcl061中均能表达,表达受苯乙酸诱导。     

巨大芽孢杆菌α-淀粉酶基因核苷酸序列分析

巨大芽孢杆菌（Ｂａｃｉｌｕｓｍｅｇａｔｅｒｉｕｍ）ＡＳ１．１２７的淀粉酶基因的全碱基序列已被测定。结构基因由１９８２ｂｐ的单一开读框架组成。由ＤＮＡ序列推测出的前体酶蛋白由６５９个氨基酸组成,Ｎ端３３个氨基酸为信号肽。成熟酶分子由６２６个氨基酸组成,分子量为６８６７６ｋＤ。该淀粉酶属糖化型α淀粉酶。并与枯草杆菌（Ｂ．ｓｕｂｔｉｌｉｓ）１６８产生的糖化型α淀粉酶之间有８３３％的同源性。分析发现两种菌产生的酶分子的Ｎ端３／４的同源性为９０４％,而Ｃ端１／４的同源性只有７０％。序列排比结果说明在淀粉酶基因的趋异进化过程中,基因突变和遗传重组都曾起过作用。     

Characterisation of mutations in GroES that allow GroEL to function as a single ring

The chaperonin GroEL contains two seven-subunit rings, and allosteric signals between them are required to complete the GroEL reaction cycle. For this reason SR1, a mutant of GroEL that forms only single rings, cannot function as a chaperone. Mutations in SR1 that restore chaperone function weaken its interaction with the cochaperonin GroES. We predicted that GroES mutants with reduced affinity for GroEL would also restore function to SR1. To test this, we mutated residues in GroES in and near its contact site with GroEL. Nearly half of the mutants showed partial function with SR1. Two mutants were confirmed to have reduced affinity for GroEL. Intriguingly, some GroES mutants were able to function with active single ring mutants of GroEL.     

A biochemical screen for GroEL/GroES inhibitors

High-throughput screening of 700,000 small molecules has identified 235 inhibitors of the GroEL/GroES-mediated refolding cycle. Dose–response analysis of a subset of these hits revealed that 21 compounds are potent inhibitors of GroEL/GroES-mediated refolding (IC₅₀ <10 μM). The screening results presented herein represent the first steps in a broader aim of developing molecular probes to study chaperonin biochemistry and physiology.     

GroEL分子伴侣研究进展

大肠杆菌的GroEL是同型寡聚复合体,由14个相对分子质量58×103亚基组成背靠背的双环结构。它在新生蛋白质的正确折叠和组装以及在热或化学逆境下变性蛋白质的恢复过程中起重要作用。同时,在大肠杆菌的跨膜转运及插入细胞质膜方面都起重要作用。这些活动依赖于GroEL与底物蛋白的疏水片断的相互作用。综述了Hsp60分子伴侣系统中研究得比较清楚的GroEL的晶体结构、功能及作用机理等方面的研究进展。     

巨大芽孢杆菌青霉素G酰化酶基因在枯草杆菌中的高表达

用ＰＣＲ方法从巨大芽孢杆菌的基因组ＤＮＡ中扩增到青霉素Ｇ酰化酶基因,并装载到枯草杆菌质粒ｐＰＺＷ１０３中,将其转化到枯草杆菌ＤＢ１０４中进行了分泌表达,重组菌株产酶无需苯乙酸诱导。在３７℃培养２４ｈ,菌液酶活力可达６ｕ／ｍｌ。１０天的连续传代实验表明重组菌株的稳定性很高。     

GroEL/GroES: structure and function of a two-stroke folding machine

Recent structural and functional studies have greatly advanced our understanding of the mechanism by which chaperonins (Cpn60) mediate protein folding, the final step in the accurate expression of genetic information. Escherichia coli GroEL has a symmetric double-toroid architecture, which binds nonnative polypeptide substrates on the hydrophobic walls of its central cavity. The asymmetric binding of ATP and cochaperonin GroES to GroEL triggers a major conformational change in the cis ring, creating an enlarged chamber into which the bound nonnative polypeptide is released. The structural changes that create the cis assembly also change the lining of the cavity wall from hydrophobic to hydrophilic, conducive to folding into the native state. ATP hydrolysis in the cis ring weakens it and primes the release of products. When ATP and GroES bind to the trans ring, it forms a stronger assembly, which disassembles the cis complex through negative cooperativity between rings. The opposing function of the two rings operates as if the system had two cylinders, one expelling the products of the reaction as the other loads up the reactants. One cycle of the reaction gives the polypeptide about 15 s to fold at the cost of seven ATP molecules. For some proteins, several cycles of GroEL assistance may be needed in order to achieve their native states.     

苏云金杆菌以色列亚种几丁质酶基因的克隆及序列分析

从苏云金芽孢杆菌以色列亚种（Bacillus thuringiensis subsp israelensis)中提取基因组DNA,通过合成1对特异性引物,用touchdown PCR的方法扩增几丁质酶ichi基因序列（GenBank登录号：AF526379）。ichi序列全长为2570bp,含有1个2067bp的开放阅读框（ORF）,编码688个氨基酸,推测分子量为75.79kDa,等电点pI=5.90的几丁质酶前体。序列和结构比较分析表明：Ichi氨基酸序列与蜡状芽孢杆菌（Bacillus cereus)28-9几丁质酶CW、蜡状芽孢杆菌CH几丁质酶B及苏云金芽孢杆菌墨西哥亚种几丁质酶的同源性分别为97.24%、97.18%、97.63%,而与苏云金芽孢杆菌巴基斯坦亚种的同源性只有63.07%。Ichi编码区由分泌信号肽（46AA）、催化区（105AA)、粘蛋白Ⅲ型同源区（74AA）及几丁质结合区（40AA）组成。     

Isolation and characterisation of mutants of GroEL that are fully functional as single rings

A key aspect of the reaction mechanism for the molecular chaperone GroEL is the transmission of an allosteric signal between the two rings of the GroEL complex. Thus, the single-ring mutant SR1 is unable to act as a chaperone as it cannot release bound substrate or GroES. We used a simple selection procedure to identify mutants of SR1 that restored chaperone activity in vivo. A large number of single amino acid changes, mapping at diverse positions throughout the protein, enabled SR1 to regain its ability to act as a chaperone while remaining as a single ring. In vivo assays were used to identify the proteins that had regained maximal activity. In some cases, no difference could be detected between strains expressing wild-type GroEL and those expressing the mutated proteins. Three of the most active proteins where the mutations were in distinct parts of the protein were purified to homogeneity and characterised in vitro. All were capable of acting efficiently as chaperones for two different GroES-dependent substrates. All three proteins bound nucleotide as effectively as did GroEL, but the binding of GroES in the presence of ATP or ADP was reduced significantly relative to the wild-type. These active single rings should provide a useful tool for studying the nature of the allosteric changes that occur in the GroEL reaction cycle.     

地衣芽孢杆菌2709碱性蛋白酶基因的克隆、序列测定及其表达

以克隆的地衣芽孢杆菌2709碱性蛋白酶编码序列的PCR扩增片段为探针。通过原位杂交从2709基因文库中筛选出两个含有完整的2709碱性蛋白酶基因的阳性克隆：Psci和Psc7。对Psc7中的插入片段构建若干亚克隆后测定了其全部DNA序列,结果显示该插入片段含2709碱性蛋白酶及其信号肽与导肽(Pro—peptide)在内的全部编码序列(1140碱基对)及长度分别为299和832碱基对的上、下游序列,该序列同M．Jacobs等克隆的地衣芽孢杆菌NcIB 6816的subtlisin Carlsberg基因序列显示了极高的同源性。通过枯草杆菌-大肠杆菌穿梭质粒Pbe2将克隆的2709碱性蛋白酶基因转入到蛋白酶缺陷型的枯草芽孢杆菌DB104中,结果表明2709碱性蛋白酶基因在枯草芽孢杆菌中得到了明显的表达。