用DREAM技术进行全长质粒快速定点突变

利用“设计限制酶辅助突变”(Designed Restriction Enzyme Assisted Mutagenesis, DREAM)进行全长质粒快速定点突变。根据突变位点附近氨基酸靶序列, 以简并密码子进行逆向推导, 这样在不改变氨基酸序列的前提下可以得到数目巨大的隐性突变体(Silent mutants), 这些突变体中包含大量的限制性酶切位点, 选择合适的酶切位点设计引物, 用Phusion超保真DNA聚合酶扩增全长质粒的DNA序列, 得到的PCR产物用T4多聚核苷酸激酶添加5￠磷酸基团后进行平末端连接, 转化大肠杆菌受体菌后用设计的酶切位点进行快速筛选。本研究用该方法成功地纠正了长约8 kb的质粒pcDNA3.1-pIgR中的突变碱基, 从而获得了多聚免疫球蛋白受体(pIgR)的野生型氨基酸序列。以上结果表明: 利用DREAM技术将限制性酶切位点引入目的基因而不改变目的蛋白质的氨基酸序列, 使突变体的筛选简单化; 配合使用高保真和高效率的Phusion DNA聚合酶可以进行长达8 kb的全长质粒的快速突变; 该方法无需使用定点突变试剂盒和特殊的受体菌, 同时避免了核酸杂交以及同位素的使用。     

快捷的定点突变效率近100%的大引物PCR方法

报道了一种新的PCR突变方法,它不需要纯化大引物或设计特别的旁侧引物．利用一个诱变引物和两个测序引物(T_m≤58℃)作为旁侧引物．第一轮PCR产物12.5 μl直接加入到50 μl的第二轮PCR反应体系作为模板和大引物,在开始第二轮PCR反应时,增加在68℃退火温度下进行10个循环的不对称PCR,这一步骤大大提高了通过600 bp或800 bp大引物所导致的突变效率．结果表明,该方法的产物能够达到高保真、97%～98%的突变效率和高产率．     

用PCR突变技术克隆艾滋病病毒蛋白酶基因

作者设计并合成了一对用于PCR技术的突变引物HIV-1 Pr1和HIV-1Pr2,分别在两引物中设计了两个突变点,使突变后基因含有EcoRI、HindⅢ和TAA序列,便于HIV-1 Pr基因的定向克隆和表达。用HIV-1 Pr1和HIV-1 Pr2作引物,采用PCR方法从HIV-1基因组DNA中扩增出了一个360bp长的DNA片段,用EcoRI和HindⅢ双酶切法将此片段定向克隆入pUC19质粒,将克隆基因插入M13mp18进行DNA序列分析。结果表明,该基因序列的读框完全正确,从而为HIV-1 Pr基因的表达及抑制剂的研究奠定了基础。     

利用Pyrobest DNA聚合酶一步法进行大片段基因的定点突变

目的:介绍一种简单、快速、高效和经济的进行大片段基因定点突变的方法。方法:小量抽提含有NM23H1-EGFP融合基因的逆转录病毒真核表达质粒pLXSN-NM23H1-EGFP,体外合成突变引物对,利用高保真Pyrobest DNA聚合酶对质粒DNA进行PCR突变反应,然后用DpnⅠ限制性内切酶消化PCR产物以去除模板DNA,取适量消化产物转化大肠杆菌XL1-Blue,随机挑选克隆进行测序筛选、鉴定所需突变株。结果:在NM23H1-EGFP基因中引入了S44A、P96S、H118F、S120G、P96S-S120G等5个替换突变位点及9bp处的插入突变。结论:该方法简单、快速、高效、经济,不须纯化中间产物,不须亚克隆,突变效率几乎为100%,是一种值得推广应用的方法。     

青霉素G酰化酶基因Ser177的定点突变

本研究用缺口以链法对肠杆菌青霉素Ｇ酰化酶（ＰＧＡ）基因Ｓｅｒ１７７进行寡核苷酸定点突变。通过ＮＩＰＡＢ（２－硝基－５－苯乙酰胺苯甲酸）试纸法筛选和测序鉴定,获得突变体Ｃｙｓ１７７,Ｇｌｙ１７７,Ａｒｇ１７７和Ａｓｎ１７７。它们的ＰＧＡ活性均已丧失。酶蛋白电泳分析表明突变体蛋白在体内正常表达。推测ＰＧＡ Ｓｅｒ１７７秀可能位于酶底物结合中心,是酶活性所必需,不能被置换。     

PCR定点突变血红蛋白基因的克隆和筛选

为开展以基因重组血红蛋白为基础的血液代用品研究,我们首先用PCR 突变法扩增出含血红蛋白α、β珠蛋白的串联基因片段,经测序表明,所克隆的α、β基因符合预期设计结果,未发生意外突变。     

利用DREAM设计和同源重组进行一步定点突变

目的：建立基于DREAM设计和同源重组的简便、快速定点突变方法。方法：设计两条包含突变的反向PCR（inverse PCR）引物,使其5'端互补从而产生同源重组,同时使用DREAM设计方案在上述引物中引入限制性内切酶位点以便突变子筛选。用能扩增长片段的高保真耐热 DNA聚合酶扩增全长的质粒DNA,直接转化大肠杆菌。转化到细菌中的全长质粒DNA PCR产物可利用其末端同源序列发生同源重组而环化。利用引入的酶切位点方便地进行突变子的筛选。结果：我们用该方法成功地对长度大于7 kb的质粒进行了定点突变。结论：本定点突变无需任何突变试剂盒和特殊的试剂,只需一步反应即可完成;利用DREAM设计使克隆筛选简便可靠,高保真耐热DNA聚合酶可保证多数突变子克隆不发生意外突变,而该酶扩增长片段的能力使该方法适合于大多数质粒不经亚克隆直接突变。     

应用聚合酶链反应突变和克隆HIV-lgag基因片段

作者设计并合成了一对突变引物PGO1和PGO2,分别在两引物中设计了两个突变点,使突变后基因含有EcoRI、BamHI和ATG及TAA序列,以便于HlV-1gag基因序列的定向克隆和表达。用PCO1和PGO2作引物,采用PCR方法从HIV-1基因组DNA中扩增出一个长504bp的DNA片段,用EcoRl和BamHI双酶切位点将此片段定向克隆入pUC19质粒。将克隆基因插入M13mp18进行DNA序列分析,结果表明,该基因序列及读框完全正确,且在其5′末端突变出EcoRI位点和ATG起始码,3′末端突变出TAA终业码和BamHI位点,从而为该基因的表达研究奠定了基础。     

PCR—单链构象多态性分析对p53基因点突变检测的研究进展

单链构象多态性（ＳＳＣＰ）对分析基因的突变是一种有效的手段,并具有其独特的优点。ＰＣＲ与ＳＳＣＰ结合后检测灵敏度更高,而在许多类型的肿瘤都存在有ｐ５３抑癌基因的突变,章综述了ＰＣＲ－ＳＳＣＰ分析技术检测ｐ５３基因突变的进展。     

Development of a technique for multiple site-directed mutagenesis of the ftf gene of Streptococcus salivarius containing palindromic sequences

Attempts at site-directed mutagenesis of the fructosyltransferase (ftf) gene of Streptococcus salivarius ATCC 25975 using standard protocols were unsuccessful and resulted in a series of deletions. These deletions appeared to commence at points within the ftf gene where there were palindromic sequences which were capable of forming closed loop structures that acted as terminators under the conditions of mutagenesis. To overcome this problem, two modified mutagenic techniques were developed. They made use of T4 DNA polymerase in conjunction with either T7 DNA polymerase at 37°C or Vent DNA polymerase from Thermococcus litoralis at an elevated temperature. These methods eliminated the need for a single-stranded DNA template and allowed polymerisation through palindromic sequences to rapidly produce multiple site-directed mutations.     

CMP羟甲基化酶MilA中与甲基羟化过程相关的关键氨基酸定点突变分析

【目的】米多霉素生物合成起始反应的MilA蛋白可以使底物胞苷酸单磷酸(CMP)的胞嘧啶碱基C5位上形成羟甲基化,形成羟甲基化胞苷酸(HmCMP),MilA是迄今发现的首个能够高效利用CMP的羟甲基化酶。MilA属于脱氧胸苷酸合成酶(TS)和脱氧胞苷酸羟甲基化酶(CH)蛋白超家族,通过三维结构预测发现它们的三维结构非常相似,CH94上的丝氨酸曾被证明与胞嘧啶上甲基变成羟甲基过程有关,MilA102上对应的氨基酸为苏氨酸,TS91上对应的氨基酸为脯氨酸。因此,突变MilA上第102位的苏氨酸,考察该氨基酸对CMP和脱氧胞苷酸(dCMP)羟甲基化反应的影响。【方法】通过定点突变技术,将MilA第102位的保守氨基酸苏氨酸(Threonine,T)分别点突变成缬氨酸(Valine,V)和亮氨酸(Leucine,L)。【结果】体外酶活实验结果显示,相对于MilA,MilA T102V对于CMP的催化活性下降87.1%,对于dCMP的催化活性没有影响;而MilA T102L均丧失了对于两种底物的活性。【结论】这表明Thr102对于MilA催化底物CMP形成HmCMP至关重要;同时,失去活性的仅比缬氨酸和野生型苏氨酸的链长多出了一个碳原子,暗示其在与底物结合时形成了一定的空间位阻效应。Thr在102位点的功能经过自然进化的微调有可能已经达到最优化。     

Investigation of the role of a second conserved serine in carboxylesterases via site-directed mutagenesis

Carboxylesterases are enzymes that catalyze the hydrolysis of ester and amide moieties. These enzymes have an active site that is composed of a nucleophile (Ser), a base (His), and an acid (Glu) that is commonly known as a catalytic triad. It has previously been observed that the majority of carboxylesterases and lipases contain a second conserved serine in their active site [Proteins, 34 (1999) 184]. To investigate whether this second serine is also involved in the catalytic mechanism, it was mutated to an alanine, a glycine or a cysteine. Site-directed mutagenesis of this conserved serine resulted in a loss of specific activity, in both the S247G and S247A mutants (5- to 15-fold), which was due to a decrease in the rate of catalysis (kcat). Due to the instability of the S247C mutant no reliable data could be attained. A carbamate inhibitor, carbaryl, was then employed to investigate whether this decrease in the kcat was due to the rate of formation of the acyl-enzyme intermediate (k2) or the rate of deacylation (k3). The S247A mutant was found only to alter k2 (2.5-fold decrease), with no effect on k3. Together with information inferred from a human carboxylesterase crystal structure, it was concluded that this serine provides an important structural support for the spatial orientation of the glutamic acid, stabilizing the catalytic triad so that it can perform the hydrolysis.     

Valine dehydrogenase from Streptomyces albus: gene cloning, heterologous expression and identification of active site by site-directed mutagenesis

A gene encoding valine dehydrogenase (Vdh) has been cloned from Streptomyces albus, a salinomycin producer, and expressed in Escherichia coli. The S. albus Vdh is composed of 364 amino acids that showed high homology with several other amino acid dehydrogenases as well as Vdhs from Streptomyces spp. and leucine and phenylalanine dehydrogenases (Ldh and Pdh) from Bacillus spp. A protein of 38 kDa, corresponding to the approximate mass of the predicted S. albus Vdh product (38.4 kDa) exhibiting specific Vdh activity, was observed when the S. albus vdh gene was overexpressed in E. coli under the controlled T7 promoter and was subsequently purified to homogeneity. Among branched- and straight-chain amino acids, L-valine and L-alpha-aminobutyrate were the preferred substrates for the enzyme. Lys-79 and Lys-91 of S. albus Vdh were highly conserved in the corresponding region of NAD(P)(+)-dependent amino acid dehydrogenase sequences. To elucidate the functional roles of the lysyl residues, the Lys residues have individually been replaced with Ala by site-directed mutagenesis. Kinetic analyses of the Lys-79 and Lys-91-mutated enzymes revealed that they are involved in the substrate binding site and catalysis, respectively, analogous to the corresponding residues in the homologous Ldh and Pdh.     

Improvement in thermostability and psychrophilicity of psychrophilic alanine racemase by site-directed mutagenesis

A psychrophilic alanine racemase from Bacillus psychrosaccharolyticus has a higher catalytic activity than a thermophilic alanine racemase from Bacillus stearothermophilus even at 60 °C in the presence of pyridoxal 5′-phosphate (PLP), although the thermostability of the former enzyme is lower than that of the latter one [FEMS Microbial. Lett. 192 (2000) 169]. In order to improve the thermostability of the psychrophilic enzyme, two hydrophilic amino acid residues (Glu150 and Arg151) at a surface loop surrounding the active site of the enzyme were substituted with the corresponding residues (Val and Ala) in the B. stearothermophilus alanine racemase. The mutant enzyme (ER150,151VA) showed a higher thermostability, and a markedly lower K_m value for PLP, than the wild type one. In addition, the catalytic activities at low temperatures and kinetic parameters of the two enzymes indicated that the mutant enzyme was more psychrophilic than the wild type one. Thus, the psychrophilic alanine racemase was improved in both psychrophilicity and thermostability by the site-directed mutagenesis. The mutant enzyme may be useful for the production of stereospecifically deuterated NADH and various -amino acids.     

Calcium-dependent protease of the cyanobacterium Anabaena: molecular cloning and expression of the gene in Escherichia coli, sequencing and site-directed mutagenesis

Summary It has been suggested that a calcium-dependent intracellular protease of the cyanobacterium, Anabaena sp., participates in the differentiation of heterocysts, cells that are specialized for fixation of N₂. Clones of the structural gene (designated prcA) for this protease from Anabaena variabilis strain ATCC 29413 and Anabaena sp. strain PCC 7120 were identified via their expression in Escherichia coli. The prcA gene from A. variabilis was sequenced. The genes of both strains, mutated by insertion of a drug resistance cassette, were returned to these same strains of Anabaena on suicide plasmids. The method of sacB-mediated positive selection for double recombinants was used to achieve replacement of the wild-type prcA genes by the mutated forms. The resulting mutants, which lacked Ca²⁺-dependent protease activity, were not impaired in heterocyst formation and grew on N₂ as sole nitrogen source.     

Identification of important residues in diketoreductase from Acinetobacter baylyi by molecular modeling and site-directed mutagenesis

Diketoreductase (DKR) from Acinetobacter baylyi exhibits a unique property of double reduction of a β, δ-diketo ester with excellent stereoselectivity, which can serve as an efficient biocatalyst for the preparation of an important chiral intermediate for cholesterol lowering statin drugs. Taken the advantage of high homology between DKR and human heart 3-hydroxyacyl-CoA dehydrogenase (HAD), a molecular model was created to compare the tertiary structures of DKR and HAD. In addition to the possible participation of His-143 in the enzyme catalysis by pH profile, three key amino acid residues, Ser-122, His-143 and Glu-155, were identified and mutated to explore the possibility of involving in the catalytic process. The catalytic activities for mutants S122A/C, H143A/K and E155Q were below detectable level, while their binding affinities to the diketo ester substrate and cofactor NADH did not change obviously. The experimental results were further supported by molecular docking, suggesting that Ser-122 and His-143 were essential for the proton transfer to the carbonyl functional groups of the substrate. Moreover, Glu-155 was crucial for maintaining the proper orientation and protonation of the imidazole ring of His-143 for efficient catalysis.     

Identification of catalytically important amino acids in human ceruloplasmin by site-directed mutagenesis

The involvement of amino acid residues previously proposed on the basis of structural data to have roles in the ferroxidase and diamine oxidase activities of human ceruloplasmin was investigated. Variants of human ceruloplasmin, in which residues proposed to be involved in electron transfer and/or iron-binding had been altered by site-directed mutagenesis, were expressed in HEK293 cells. E633A and E597A/H602A variants exhibited reduction in both activities by 50–60% compared to recombinant wild-type ceruloplasmin. The variant E935A/H940A had reduced ferroxidase activity (50%) but unaltered diamine oxidase activity, whereas the variant E971A exhibited enhanced diamine oxidase activity. For the L329M variant, both activities were identical to those of wild-type ceruloplasmin.