DNA错配修复基因mutS的高效表达及表达产物活性鉴定

将DNA错配修复基因mutS（2.56kb）克隆于分泌型原核表达载体pET32a（+）上,以N端融合6个组氨酸的形式在E.coliAD494(DE3) 中进行了IPTG诱导表达。SDSPAGE分析证实有一与预期分子量相应的诱导表达条带,其表达量占全菌蛋白质的35％左右,且表达蛋白以可溶形式存在。利用固定化金属离子（Ni²⁺）配体亲和层析柱纯化目的蛋白,其纯度为90％以上。与含有错配碱基DNA双链的结合反应证明该蛋白具有特异性识别、结合含有错配碱基DNA双链的生物活性。     

MutL融合蛋白的高效表达及其伴侣功能研究(英文)

DNA错配修复蛋白MutL和其它的修复蛋白相互作用来共同完成大肠杆菌甲基介导的错配修复过程 .为研究修复蛋白MutL的体外生物功能构建了融合蛋白Trx His6 Linkerpeptide MutL(THLL)的表达载体并使其高效表达及易于纯化 .mutL基因片段是以E .coliK 12基因组为模板经PCR扩增获得 ,并通过基因的体外拼接成功构建了融合蛋白THLL表达载体pET32a linkerpeptide mutL .重组菌株E .coliAD4 94 (DE3) pET32a linkerpeptide mutL经过IPTG的诱导表达了融合蛋白THLL .收集菌体细胞、超声波破碎后离心取上清进行SDS PAGE分析 ,结果表明有一与预期分子量(84kD)相应的诱导表达条带出现 ,其表达量约占全细胞蛋白的 30 %且以可溶形式存在 .利用固定化金属离子 (Ni2 +)配体亲和层析柱纯化融合蛋白THLL ,其纯度达到 90 % .通过非变性凝胶电泳分析 ,对融合蛋白THLL在DNA错配修复过程中的分子伴侣生物功能进行了系统研究 .结果表明 ,THLL能增加融合蛋白Trx His6 Linkerpeptide MutS (THLS)与含有错配碱基DNA双链的结合 ,但受ATP的浓度变化影响很大     

SSB蛋白的高效表达及其与ssDNA的相互作用

大肠杆菌单链结合蛋白SSB在DNA复制、重组和修复中起着重要作用。为研究单链结合蛋白SSB的体外生物功能构建了融合蛋白SSB的表达载体并使其高效表达及易于纯化。ssb基因片段是以E.coli K-12基因组为模板经PCR扩增获得,并通过基因的体外拼接成功构建了表达载体pQE30-ssb。重组菌株M15/ pQE30-ssb经过IPTG的诱导表达了蛋白SSB。收集菌体细胞、超声波破碎后离心取上清进行SDS-PAGE分析,结果表明有一与预期分子量（20.6 kD）相应的诱导表达条带出现,其表达量约占全细胞蛋白的30％且以可溶形式存在。利用固定化金属离子（Ni2+）配体亲和层析柱纯化融合蛋白SSB,其纯度达到90％。通过凝胶层析和等离子共振技术对SSB的生物功能进行了系统研究分析。结果表明,SSB蛋白以四聚体形式与单链DNA分子结合,其亲和力常数（KD）为4.79×10-7 M。     

人R_O蛋白(60kD)编码序列的PCR产物在E.coli中的克隆与表达

通过聚合酶链反应 ( PCR)自人脾 c DNA扩增 RO 蛋白 ( 60 k D)编码 DNA片段 .将该片段定向插入麦芽糖结合蛋白 ( MBP)融合系统的 p MALTM- c载体中并转化 E.coli ( DH5α) ,通过酶联免疫印迹 ( IBT)筛选出具有 RO 抗原性的阳性克隆 .绝大部分阳性克隆表达完整的 RO 融合蛋白 ( 1 0 0k D) .但在传代过程中表达水平很快降低 ;少数阳性克隆虽然表达非完整 RO 融合蛋白 (分子量 <80k D) ,但表达水平却高而且稳定 .同时保留了很强的 RO 抗原性 .产生非完整融合蛋白的一个原因是 ,PCR碱基错配引起 RO 编码 DNA的序列缺失 ;另一原因是融合蛋白在表达过程中被降解     

DNA错配修复蛋白MutS和MutL的相互作用研究

MutL 和 MutS 是DNA错配修复系统中起关键作用的修复蛋白. 利用基因融合技术高效表达了MutL 和 MutS融合蛋白,并利用它们发展了一种研究二者相互作用的简便方法. 融合蛋白MutL-GFP (Trx-His6-GFP-(Ser-Gly)6-MutL),MutL-Strep tagⅡ (Trx-His6-(Ser-Gly)6-Strep tagⅡ-(Ser-Gly)6-MutL) 和 MutS (Trx-His6-(Ser-Gly)6-MutS) 被构建并在大肠杆菌中高效表达. 收集菌体细胞、超声波破碎后离心取上清进行SDS-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 分析,结果表明有与预期分子质量相应的诱导表达条带出现,其表达量约占全细胞蛋白的30％且以可溶形式存在. 利用固定化金属离子配体亲和层析柱分别纯化融合蛋白,其纯度达到90％. 通过将MutS蛋白固定的方法研究两种MutL融合蛋白分别与MutS之间的相互作用. 结果表明：只有MutS蛋白与含有错配碱基DNA分子结合后才与MutL蛋白发生相互作用. 通过检测MutL融合蛋白标记的绿色荧光信号或酶学显色信号来鉴定相互作用的发生. 建立的融合分子系统方法也为研究其他的蛋白质或生物大分子之间的相互作用提供了一个技术平台.     

破伤风毒素C片段(TTc)在大肠杆菌中的表达、优化与鉴定

HTSS以一株破伤风生产菌株基因组DNA为模板,通过上游引物中几个碱基的修改,PCR扩增出破伤风毒素C片段(TTc)基因,构建了原核表达质粒pET-42(b)/TTc,在大肠杆菌BL21(DE3)中表达。重组蛋白分子量约50kD,表达量为22%,超声波破碎显示为可溶性重组蛋白。通过对培养基、诱导时间、诱导温度的优化,重组蛋白的表达量和可溶性均有提高。Western blotting检测表达产物可与破伤风C片段单克隆抗体产生特异的免疫反应。该工作为亚单位疫苗或载体蛋白的开发奠定了基础。     

大白菜BcPLH基因的原核表达及其蛋白产物的Western分析

BcpLH基因是大白菜包叶组织特异的新基因,含有双链RNA结合域。在含有His标记序列的原核表达载体pET28-a(+)上插入Bc-pLH基因的cDNA,在大肠杆菌BL21(DE3)中诱导表达出了特异性蛋白,并免疫大白兔制备出高效价的抗血清;同时,将BcpLH基因插入到含有超级助溶剂的pMAL-c2载体上,并在大肠杆菌DH5α中诱导表达,结果获得了可溶的蛋白。Western斑点印迹分析的结果证明了BcpLH的特异性。BcpLH活性蛋白及其抗血清的产生为研究BcpLH基因的RNA结合     

烟草花叶病毒运动蛋白cDNA的克隆及融合蛋白的表达

从烟草花叶病毒(TMV)中提取总RNA,通过反转录PCR (RTPCR) 扩增得到其运动蛋白(MP)的基因,将扩增产物克隆到pMD18T载体上。DNA序列分析表明,所得到的运动蛋白的基因全长为807bp (GenBank接受号AY300161), 与已发表TMV序列（GenBank登陆号为NC-001367）和同属的番茄花叶病毒（ToMV, GenBank登陆号为NC-002692相比核苷酸的同源性分别为98.0％和80.9％,氨基酸的同源性分别为99.1％和80.0％。 将目的片段亚克隆到表达载体pET30a上,并在大肠杆菌JM109中诱导表达,诱导9h 后,融合蛋白表达量最大。诱导后的工程菌超声后经SDSPAGE检测,融合蛋白以可溶形式存在。     

重组人IL-1ra在大肠杆菌中的可溶性表达

利用PCR技术从含有IL-1ra的质粒上扩增IL-1ra基因,经过序列测定后插入表达载体pTIG-Trx,并转化大肠杆菌BL21(DE3),用IPTG进行诱导表达。经SDS-PAGE分析显示,IL-1ra表达质粒在大肠杆菌中的诱导表达产物出现相对分子量大约为17000的一条新生蛋白质带,其大小与预期结果一致,经Western和ELISA分析,证明该带即为目的蛋白,SDS-PAGE显示目的蛋白全部以可溶性蛋白的形式存在。超声破碎后,上清经金属螯和层析纯化获得纯度约为98％的蛋白样品。     

异双聚体电泳及其在遗传学差异筛选中的应用

介绍了一种灵敏、简便、快速测定双链PCR产物或DNA片断单个碱基差异的方法。该法借助常规PAGE电泳,能区分出有单个碱基错配而发生构型改变的异双聚体与碱基互补配对的同源双聚体双链DNA分子;并判断出序列中碱基错配的百分率。     

Alpha-shaped DNA loops induced by MutS

DNA mismatch repair (MMR) is critical for the maintenance of genomic stability. MMR is initiated by recognition of DNA mismatches by the protein, MutS, which subsequently recruits downstream repair factors. To better understand the mechanism by which MutS identifies and specifically binds mismatched basepairs embedded in random DNA sequences, we monitored the interaction between MutS and DNA substrates using atomic force microscopy (AFM). An α-shaped DNA loop formed by the interaction between MutS and DNA, which was independent of whether or not a mismatch was present in the DNA substrate. These data indicate that MutS associates with DNA non-specifically and forms an α-loop interaction with the DNA substrate. In this conformation, MutS is able to scan two arms of DNA simultaneously for each MutS dimer formed.     

A single mismatch in the DNA induces enhanced aggregation of MutS. Hydrodynamic analyses of the protein-DNA complexes

Changes in the oligomeric status of MutS protein was probed in solution by dynamic light scattering (DLS), and corroborated by sedimentation analyses. In the absence of any nucleotide cofactor, free MutS protein [hydrodynamic radius (Rh) of 10-12 nm] shows a small increment in size (Rh 14 nm) following the addition of homoduplex DNA (121 bp), whereas the same increases to about 18-20 nm with heteroduplex DNA containing a mismatch. MutS forms large aggregates (Rh > 500 nm) with ATP, but not in the presence of a poorly hydrolysable analogue of ATP (ATPgammaS). Addition of either homo- or heteroduplex DNA attenuates the same, due to protein recruitment to DNA. However, the same protein/DNA complexes, at high concentration of ATP (10 mm), manifest an interesting property where the presence of a single mismatch provokes a much larger oligomerization of MutS on DNA (Rh > 500 nm in the presence of MutL) as compared to the normal homoduplex (Rh approximately 100-200 nm) and such mismatch induced MutS aggregation is entirely sustained by the ongoing hydrolysis of ATP in the reaction. We speculate that the surprising property of a single mismatch, in nucleating a massive aggregation of MutS encompassing the bound DNA might play an important role in mismatch repair system.     

The Phe-X-Glu DNA binding motif of MutS. The role of hydrogen bonding in mismatch recognition

The crystal structures of MutS protein from Thermus aquaticus and Escherichia coli in a complex with a mismatch-containing DNA duplex reveal that the Glu residue in a conserved Phe-X-Glu motif participates in a hydrogen-bonded contact with either an unpaired thymidine or the thymidine of a G-T base-base mismatch. Here, the role of hydrogen bonding in mismatch recognition by MutS is assessed. The relative affinities of MutS for DNA duplexes containing nonpolar shape mimics of A and T, 4-methylbenzimidazole (Z), and difluorotoluene (F), respectively, that lack hydrogen bonding donors and acceptors, are determined in gel mobility shift assays. The results provide support for an induced fit mode of mismatch binding in which duplexes destabilized by mismatches are preferred substrates for kinking by MutS. Hydrogen bonding between the O epsilon 2 group of Glu and the mismatched base contributes only marginally to mismatch recognition and is significantly less important than the aromatic ring stack with the conserved Phe residue. A MutS protein in which Ala is substituted for Glu(38) is shown to be defective for mismatch repair in vivo. DNA binding studies reveal a novel role for the conserved Glu residue in the establishment of mismatch discrimination by MutS.     

Slow Conformational Changes in MutS and DNA Direct Ordered Transitions between Mismatch Search,Recognition and Signaling of DNA Repair

MutS functions in mismatch repair (MMR) to scan DNA for errors, identify a target site and trigger subsequent events in the pathway leading to error removal and DNA re-synthesis. These actions, enabled by the ATPase activity of MutS, are now beginning to be analyzed from the perspective of the protein itself. This study provides the first ensemble transient kinetic data on MutS conformational dynamics as it works with DNA and ATP in MMR. Using a combination of fluorescence probes (on Thermus aquaticus MutS and DNA) and signals (intensity, anisotropy and resonance energy transfer), we have monitored the timing of key conformational changes in MutS that are coupled to mismatch binding and recognition, ATP binding and hydrolysis, as well as sliding clamp formation and signaling of repair. Significant findings include (a) a slow step that follows weak initial interaction between MutS and DNA, in which concerted conformational changes in both macromolecules control mismatch recognition, and (b) rapid, binary switching of MutS conformations that is concerted with ATP binding and hydrolysis and (c) is stalled after mismatch recognition to control formation of the ATP-bound MutS sliding clamp. These rate-limiting pre- and post-mismatch recognition events outline the mechanism of action of MutS on DNA during initiation of MMR.     

The construction of bifunctional fusion proteins consisting of MutS and GFP

MutS as a mismatch binding protein is a promising tool for SNP detection. Green fluorescent protein (GFP) is known as an excellent reporter domain. We constructed chimeric proteins consisting of MutS from Thermus thermophilus and GFPuv from Aequorea victoria by cloning the GFPuv gene into the plasmid vectors carrying the mutS gene. The GFPuv domain fused to the N-terminus of MutS (histag-GFP-MutS) exhibited the same level of green fluorescence as free GFPuv. To obtain the fluorescing histag-GFP-MutS protein the expression at 30 degrees C was required, while free GFPuv fluoresces when expressed both at 30 and 37 degrees C. The chimeric protein where the GFPuv domain was fused to the C-terminus of MutS exhibited much weaker green fluorescence (20-25% compared with those of histag-GFP-MutS or free GFPuv). The insertion of (ProGly)5 peptide linker between the MutS and GFP domains resulted in no significant improvement in GFP fluorescence. No shifts in the excitation and emission spectra have been observed for the GFP domain in the fusion proteins. The fusion proteins with GFP at the N- and C-terminus of MutS recognised DNA mismatches similarly like T. thermophilus MutS. The fluorescent proteins recognising DNA mismatches could be useful for SNP scanning or intracellular DNA analysis. The fusion proteins around 125 kDa were efficiently expressed in E. coli and purified in milligram amounts using metal chellate affinity chromatography.     

Disruption of the helix-u-turn-helix motif of MutS protein: loss of subunit dimerization, mismatch binding and ATP hydrolysis

The DNA mismatch repair protein, MutS, is a dimeric protein that recognizes mismatched bases and has an intrinsic ATPase activity. Here, a series of Taq MutS proteins having C-terminal truncations in the vicinity of a highly conserved helix-u-turn-helix (HuH) motif are assessed for subunit oligomerization, ATPase activity and DNA mismatch binding. Those proteins containing an intact HuH region are dimers; those without the HuH region are predominantly monomers in solution. Steady-state kinetics of truncated but dimeric MutS proteins reveals only modest decreases in their ATPase activity compared to full-length protein. In contrast, disruption of the HuH region results in a greatly attenuated ATPase activity. In addition, only dimeric MutS proteins are proficient for mismatch binding. Finally, an analysis of the mismatch repair competency of truncated Escherichia coli MutS proteins in a rifampicin mutator assay confirms that the HuH region is critical for in vivo function. These findings indicate that dimerization is critical for both the ATPase and DNA mismatch binding activities of MutS, and corroborate several key features of the MutS structure recently deduced from X-ray crystallographic studies.     

Impact of mutS inactivation on foreign DNA acquisition by natural transformation in Pseudomonas stutzeri

In prokaryotic mismatch repair the MutS protein and its homologs recognize the mismatches. The mutS gene of naturally transformable Pseudomonas stutzeri ATCC 17587 (genomovar 2) was identified and characterized. The deduced amino acid sequence (859 amino acids; 95.6 kDa) displayed protein domains I to IV and a mismatch-binding motif similar to those in MutS of Escherichia coli. A mutS::aac mutant showed 20- to 163-fold-greater spontaneous mutability. Transformation experiments with DNA fragments of rpoB containing single nucleotide changes (providing rifampin resistance) indicated that mismatches resulting from both transitions and transversions were eliminated with about 90% efficiency in mutS+. The mutS+ gene of strain ATCC 17587 did not complement an E. coli mutant but partially complemented a P. stutzeri JM300 mutant (genomovar 4). The declining heterogamic transformation by DNA with 0.1 to 14.6% sequence divergence was partially alleviated by mutS::aac, indicating that there was a 14 to 16% contribution of mismatch repair to sexual isolation. Expression of mutS+ from a multicopy plasmid eliminated autogamic transformation and greatly decreased heterogamic transformation, suggesting that there is strong limitation of MutS in the wild type for marker rejection. Remarkably, mutS::aac altered foreign DNA acquisition by homology-facilitated illegitimate recombination (HFIR) during transformation, as follows: (i) the mean length of acquired DNA was increased in transformants having a net gain of DNA, (ii) the HFIR events became clustered (hot spots) and less dependent on microhomologies, which may have been due to topoisomerase action, and (iii) a novel type of transformants (14%) had integrated foreign DNA with no loss of resident DNA. We concluded that in P. stutzeri upregulation of MutS could enforce sexual isolation and downregulation could increase foreign DNA acquisition and that MutS affects mechanisms of HFIR.     

The effects of nucleotides on MutS-DNA binding kinetics clarify the role of MutS ATPase activity in mismatch repair

MutS protein initiates mismatch repair with recognition of a non-Watson-Crick base-pair or base insertion/deletion site in DNA, and its interactions with DNA are modulated by ATPase activity. Here, we present a kinetic analysis of these interactions, including the effects of ATP binding and hydrolysis, reported directly from the mismatch site by 2-aminopurine fluorescence. When free of nucleotides, the Thermus aquaticus MutS dimer binds a mismatch rapidly (k(ON)=3 x 10(6) M(-1) s(-1)) and forms a stable complex with a half-life of 10 s (k(OFF)=0.07 s(-1)). When one or both nucleotide-binding sites on the MutS*mismatch complex are occupied by ATP, the complex remains fairly stable, with a half-life of 5-7 s (k(OFF)=0.1-0.14 s(-1)), although MutS(ATP) becomes incapable of (re-)binding the mismatch. When one or both nucleotide-binding sites on the MutS dimer are occupied by ADP, the MutS*mismatch complex forms rapidly (k(ON)=7.3 x 10(6) M(-1) s(-1)) and also dissociates rapidly, with a half-life of 0.4 s (k(OFF)=1.7 s(-1)). Integration of these MutS DNA-binding kinetics with previously described ATPase kinetics reveals that: (a) in the absence of a mismatch, MutS in the ADP-bound form engages in highly dynamic interactions with DNA, perhaps probing base-pairs for errors; (b) in the presence of a mismatch, MutS stabilized in the ATP-bound form releases the mismatch slowly, perhaps allowing for onsite interactions with downstream repair proteins; (c) ATP-bound MutS then moves off the mismatch, perhaps as a mobile clamp facilitating repair reactions at distant sites on DNA, until ATP is hydrolyzed (or dissociates) and the protein turns over.     

Composite active site of an ABC ATPase: MutS uses ATP to verify mismatch recognition and authorize DNA repair

The MutS protein initiates DNA mismatch repair by recognizing mispaired and unpaired bases embedded in duplex DNA and activating endo- and exonucleases to remove the mismatch. Members of the MutS family also possess a conserved ATPase activity that belongs to the ATP binding cassette (ABC) superfamily. Here we report the crystal structure of a ternary complex of MutS-DNA-ADP and assays of initiation of mismatch repair in conjunction with perturbation of the composite ATPase active site by mutagenesis. These studies indicate that MutS has to bind both ATP and the mismatch DNA simultaneously in order to activate the other mismatch repair proteins. We propose that the MutS ATPase activity plays a proofreading role in DNA mismatch repair, verification of mismatch recognition, and authorization of repair.