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。     

Biophysical and kinetic analysis of wild-type and site-directed mutants of the isolated and native dehydroquinate synthase domain of the AROM protein

Dehydroquinate synthase (DHQS) is the N-terminal domain of the pentafunctional AROM protein that catalyses steps 2 to 7 in the shikimate pathway in microbial eukaryotes. DHQS converts 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) to dehydroquinate in a reaction that includes alcohol oxidation, phosphate beta-elimination, carbonyl reduction, ring opening, and intramolecular aldol condensation. Kinetic analysis of the isolated DHQS domains with the AROM protein showed that for the substrate DAHP the difference in Km is less than a factor of 3, that the turnover numbers differed by 24%, and that the Km for NAD+ differs by a factor of 3. Isothermal titration calorimetry revealed that a second (inhibitory) site for divalent metal binding has an approximately 4000-fold increase in KD compared to the catalytic binding site. Inhibitor studies have suggested the enzyme could act as a simple oxidoreductase with several of the reactions occurring spontaneously, whereas structural studies have implied that DHQS participates in all steps of the reaction. Analysis of site-directed mutants experimentally test and support this latter hypothesis. Differential scanning calorimetry, circular dichroism spectroscopy, and molecular exclusion chromatography demonstrate that the mutant DHQS retain their secondary and quaternary structures and their ligand binding capacity. R130K has a 135-fold reduction in specific activity with DAHP and a greater than 1100-fold decrease in the kcat/Km ratio, whereas R130A is inactive.     

Improving the binding affinity of an antibody using molecular modeling and site-directed mutagenesis

Activated Factor X releases F1.2, a 271-amino acid peptide, from the amino terminus of prothrombin during blood coagulation. A nine-amino acid peptide, C9 (DSDRAIEGR), corresponding to the carboxyl terminus of F1.2 was synthesized and used to produce a monoclonal antibody, TA1 (K(D)) 1.22 x 10(-6) M). To model the TA1 antibody, we entered the sequence information of the cloned TA1 Fv into the antibody modeling program, ABM, which combines homology methods, conformational search procedures, and energy screening and has proved to be a reliable and reproducible antibody modeling method. Using a novel protein fusion procedure, we expressed the C9 peptide fused to the carboxyl terminus of the PENI repressor protein from Bacillus licheniformis in Escherichia coli. We constructed fusion proteins containing alanine substitutions for each amino acid in the C9 epitope. Binding studies, using the C9 alanine mutants and TA1, and spatial constraints predicted by the modeled TA1 binding cleft enabled us to establish a plausible conformation for C9 complexed with TA1. Furthermore, based on binding results of conservative amino acid substitutions in C9 and mutations in the antibody, we were able to refine the complex model and identify antibody mutations that would improve binding affinity.     

Localization by site-directed mutagenesis of a galantamine binding site on α7 nicotinic acetylcholine receptor extracellular domain

Galantamine is an approved drug treatment for Alzheimer’s disease. Initially identified as a weak cholinesterase inhibitor, we have established that galantamine mainly acts as an ‘allosterically potentiating ligand (APL)’ of nicotinic acetylcholine receptors (nAChR). Meanwhile other ‘positive allosteric modulators (PAM)’ of nAChR channel activity have been discovered, and for one of them a binding site within the transmembrane domain has been proposed. Here we show, by performing site-directed mutagenesis studies of ectopically expressed chimeric chicken α7/mouse 5-hydroxytryptamine 3 receptor-channel complex, in combination with whole-cell current measurements, in the presence and absence of galantamine, that the APL binding site is different from the proposed PAM binding site. We demonstrate that residues T197, I196, and F198 of ß-strand 10 represent major elements of the galantamine binding site. Residue K123, earlier suggested as being ‘close to’ the APL binding site, is not part of this site but rather appears to play a role in coupling of agonist binding to channel opening and closing. Our data confirm our earlier results that the galantamine binding site is different from the ACh binding site. Both sites are in close proximity and hence may influence each other in a synergistic fashion. Other interesting areas identified in the present study are a ‘hinge’ region around and containing residues F122, K123, and K143 possibly being involved in relaying the signal of agonist binding to gating of the transmembrane channel, and a ‘folding centre’, with P119 as the dominating residue, that crucially positions the agonist binding site with respect to the hinge region.     

定点突变改造提高纺锤形赖氨酸芽孢杆菌氨基甲酸乙酯水解酶稳定性

氨基甲酸乙酯(Ethyl carbamate,EC)是一种存在于发酵食品和酿造酒精饮料中的潜在致癌物质。利用生物酶法去除食品饮料中的EC是一种较为安全有效的方法。本研究以来源于赖氨酸芽孢杆菌Lysinibacillus fusiformis SC02的氨基甲酸乙酯水解酶为研究对象,采用计算机辅助设计突变位点,构建了其不稳定区域Q328位点的饱和突变体。通过酶学性质分析发现,突变体Q328C和Q328V在40℃下的半衰期分别提高了7.46和1.99倍,Q328R在高温下也有比原酶更好的耐受性。此外,突变体Q328C对乙醇的耐受性和酸耐受性也有所提高。对氨基甲酸乙酯水解酶分子改造的结果表明,通过改造其不稳定区域Q328位点,可以提高酶的热稳定性及对酸和乙醇的耐受性。     

Mutational fitness effects in RNA and single-stranded DNA viruses: common patterns revealed by site-directed mutagenesis studies

The fitness effects of mutations are central to evolution, yet have begun to be characterized in detail only recently. Site-directed mutagenesis is a powerful tool for achieving this goal, which is particularly suited for viruses because of their small genomes. Here, I discuss the evolutionary relevance of mutational fitness effects and critically review previous site-directed mutagenesis studies. The effects of single-nucleotide substitutions are standardized and compared for five RNA or single-stranded DNA viruses infecting bacteria, plants or animals. All viruses examined show very low tolerance to mutation when compared with cellular organisms. Moreover, for non-lethal mutations, the mean fitness reduction caused by single mutations is remarkably constant (0.10–0.13), whereas the fraction of lethals varies only modestly (0.20–0.41). Other summary statistics are provided. These generalizations about the distribution of mutational fitness effects can help us to better understand the evolution of RNA and single-stranded DNA viruses.     

The role of an active-site lysyl residue of spinach phosphoribulokinase as explored by site-directed mutagenesis

Based on selective labeling by ATP analogues, Lys68 of the Calvin Cycle enzyme phosphoribulokinase (PRK) from spinach has been assigned to the active-site region [Miziorkoet al. (1990),J. Biol. Chem. 265, 3642–3647]. The equivalent position is occupied by lysyl or arginyl residues in the PRK from both prokaryotic and eukaryotic sources, suggesting a requirement for a basic residue at this location. To examine this possibility, we have replaced Lys68 of the spinach enzyme with arginyl, glutaminyl, alanyl, or glutamyl residues by site-directed mutagenesis. All of the mutant enzymes retain substantial kinase activity; and even in the case of the radical substitution by glutamate, theK _m values for ATP and ribulose 5-phosphate are not perturbed significantly. Glutamate at position-68 may destabilize tertiary structure, because the yield of this mutant protein from transformedE. coli is quite low compared to that of the other proteins in this series. Despite the active-site proximity of Lys68, our results show that this residue does not play a key role in catalysis or substrate binding.     

Mapping the antigenic determinants and reducing the immunogenicity of trichosanthin by site-directed mutagenesis

Summary Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) possessing multiple pharmacological properties. One of its interesting properties is to inhibit human immunodeficiency virus (HIV) replication but its strong immunogenicity has limited the repeated clinical administration. To map the antigenic determinants and reduce the immunogenicity of TCS, two potential antigenic sites (YFF81–83 and KR173–174) were identified by computer modeling, and then three TCS mutants namely TCS_{YFF81–83ACS}, TCS_{KR173–174CG}, and TCS_YFF-KR were constructed by site-directed mutagenesis. The RI activity and DNase-like activity of the three constructed TCS mutants were similar to natural TCS but with much lower immunogenicity. Results suggested that the two selected sites are all located at or near the antigenic determinants of TCS. In toxicity studies, the LD₅₀ of the three TCS mutants was not different from natural TCS. These findings would be useful in designing a better therapeutic agent for AIDS.Qunxing An and Sanhua Wei equally contribute to this work.     

利用定点突变法研究精氨酸脱亚胺酶活性的影响机制

精氨酸脱亚胺酶(ADI)是一种针对精氨酸缺陷型癌症(如:肝癌、黑素瘤)的新药,目前处于临床三期试验。文中通过定点突变技术分析了精氨酸脱亚胺酶的特定氨基酸位点对酶活力的影响机制。针对已报道的关键氨基酸残基A128、H404、I410,采用QuikChange法进行定点突变,获得ADI突变株M1(A128T)、M2(H404R)、M3(I410L)和M4(A128T/H404R)。将突变株在大肠杆菌BL21(DE3)中进行重组表达,并对纯化获得的突变蛋白进行酶学性质研究。结果表明,突变位点A128T和H404R对ADI最适pH的提高,生理中性(pH 7.4)条件下的酶活力和稳定性的提高,以及Km值的降低均具有显著的作用。研究结果为阐明ADI的酶活力影响机制和蛋白质的理性改造提供了一定的依据。     

Conferring the binding properties of the mouse MHC class I-related receptor, FcRn, onto the human ortholog by sequential rounds of site-directed mutagenesis

The MHC class I-related receptor, FcRn, is involved in binding and transporting immunoglobulin G (IgG) within and across cells. In contrast to mouse FcRn, which binds to IgGs from multiple different species, human FcRn is surprisingly stringent in binding specificity. For example, human FcRn does not bind to mouse IgG1 or IgG2a and interacts only weakly with mouse IgG2b. Here, we have used site-directed mutagenesis in combination with interaction (surface plasmon resonance) studies, with the goal of generating human FcRn variants that more closely resemble mouse FcRn in binding specificity. Our studies show that residues encompassing and extending away from the interaction site on the alpha2 helix of FcRn play a significant and most likely indirect role in FcRn-IgG interactions. Further, by combining mutations in the alpha2 helix with those in a non-conserved region of the alpha1 helix encompassing residues 79-89, we have generated a human FcRn variant that has properties very similar to those of mouse FcRn. These studies define the molecular basis for the marked difference in binding specificity between human and rodent FcRn, and give insight into how human FcRn recognizes IgGs.     

定点突变提高苯丙氨酸羟化酶的热稳定性

嗜热菌中,蛋白质存在Ala替换Gly以及Arg替换Lys的趋势。为了提高紫色色杆菌来源的苯丙氨酸羟化酶的热稳定性,将该酶中所有Gly突变成Ala,Lys突变成Arg,筛选获得热稳定性提高的突变体,并进行组合突变,对突变酶的酶学性质进行研究。结果表明,突变酶K94R和G221A在50℃的半衰期分别为26.2 min、16.8 min,比原始酶(9.0 min)分别提高了1.9倍、0.9倍,同时组合突变酶K94R/G221A在50℃处理1 h后仍保留65.6%的酶活,比原始酶(8.6%)高出6.6倍。圆二色谱结果显示原始酶和突变酶K94R、G221A及K94R/G221A的T_m值分别为51.5℃、53.8℃、53.1℃和54.8℃。蛋白三维结构模拟推测突变体热稳定性提高机理为:突变体K94R中Arg94与Ile95之间形成额外氢键,稳定其所在的柔性区域;突变体G221A中Ala221与Leu281产生疏水作用,稳定酶分子C-端柔性区。该研究结果为蛋白质热稳定性改造提供了参考,也为苯丙氨酸羟化酶在功能性食品领域的应用奠定了基础。     

Anti-oxidative effect of ribonuclease inhibitor by site-directed mutagenesis and expression in Pichia pastoris

Human placental ribonuclease inhibitor(hRI)is an acidic protein of Mr-50kDa with unusually high contents of leucine and cysteine residues.It is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonuclease.hRI has 32 cysteine residues,and the oxidative formation of disulfide bonds from those cysteine residues is a rapid cooperative process that inactivates hRI.The most proximal cysteine residues in native hRI are two pairs that are adjacent in sequence.In the present aork,two molecules of alanine substituting for Cys328 and Cys329 were performed by site-directed mutagenesis.The site-mutated RI cDNA was constructed into plasmid pPIC9K and then transformed Pichia pastoris GS115 by electroporation.After colony screening,the bacterium was cultured and the product Was purified with affinity chromatography.The affinity of the recombinant human RI with double site mutation was examined for RNase A and its anti-oxidative effect.Results indicated that there were not many changes in the affinity for RNase A detected when compared with the wild type of RI.But the capacity of anti-oxidative effect increased by 7～9 times.The enhancement in anti-oxidative efrect might be attributed to preventing the formation of disulfide bond between Cys328 and Cys329 and the three dimensional structure of RI was thereby maintained.     

普鲁兰酶基因在毕赤酵母中组成型表达及定点突变研究

合成Bacillus acidopullulyticus的全长普鲁兰酶基因并在毕赤酵母X-33中进行组成型外分泌表达,重组酶的最适作用温度为60℃,最适作用pH值为4.5~5.0,酶比活力为2.0 U/mg.采用重叠延伸PCR方法对普鲁兰酶基因进行定点突变,实验结果表明,625、626位点Ala、Leu氨基酸突变为Leu、Tyr氨基酸后,该酶的催化效率有所降低,而Gln487Ala的突变对催化效率没有较大的影响.该研究结果为探究关键氨基酸区域对催化效率的影响提供了一定的理论和实验基础.     

定点突变提高土曲霉Aspergillus terreus脂肪酶的催化活性

本研究旨在利用理性设计的方法来提高来源于土曲霉Aspergillus terreus的酸性脂肪酶ATL的催化活力。通过同源比对,选择脂肪酶盖子区域和底物结合口袋域中的位点进行定点突变,得到8种ATL的突变脂肪酶。结果发现,盖子区域突变酶ATLLid与底物结合口袋域突变酶ATLV218W的催化活性显著提高。ATLLid和ATLV218W对底物对硝基苯酚月桂酸酯p-nitrophenyl laurate(p-NPL)的催化活性最高,k_(cat)值较ATL分别提高了39.37倍和50.79倍,k_(cat)/K_m值较ATL分别提高了2.85倍和8.48倍。与ATL相比,ATLLid和ATLV218W的热稳定性略有下降,最适p H为5.0,p H 4.0–8.0具有较好的稳定性,说明突变未对ATL的嗜酸耐酸特性产生影响。通过同源建模模拟及分子对接技术分析底物p-NPL与酶分子间的相互作用,解析了ATLLid和ATLV218W催化活性提高的机理。     

Expression improvement and mechanistic study of the retro-Diels-Alderase catalytic antibody 10F11 by site-directed mutagenesis

Antibody 10F11 catalyzes the retro-Diels-Alder reaction of the bicyclic prodrug 1 releasing HNO and anthracene 4 (kcat/kuncat=2500). Earlier X-ray crystal structures of Fab 10F11 showed that tryptophan H104 at the bottom of the binding pocket interacts by pi-stacking with the aromatic ring of the substrate. Antibody 10F11 was expressed as a chimeric Fab and subjected to site-directed mutagenesis. Expression was improved by substituting a serine for a phenylalanine residue on the Fv-domain surface. Nine active-site mutants were then prepared including replacements at TrpH104, PheH101 and SerH100. Catalysis depends mainly on TrpH104 and PheH101. Catalysis is most likely caused by a combination of shape complementarity and specific electronic interactions between transition state and the aromatic residue H104. Medium and de-solvation effects have no effect on the reaction rate. Catalysis was improved to (kcat/kuncat=6300) by substituting phenylalanine for LeuL101 to indirectly enhance pi-stacking between transition state and TrpH104.     

Anti-oxidative effect of ribonuclease inhibitor by site-directed mutagenesis and expression in Pichia pastoris

Human placental ribonuclease inhibitor (hRI) is an acidic protein of Mr∼50kDa with unusually high contents of leucine and cysteine residues. It is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonuclease. hRI has 32 cysteine residues, and the oxidative formation of disulfide bonds from those cysteine residues is a rapid cooperative process that inactivates hRI. The most proximal cysteine residues in native hRI are two pairs that are adjacent in sequence. In the present aork, two molecules of alanine substituting for Cys328 and Cys329 were performed by site-directed mutagenesis. The site-mutated RI cDNA was constructed into plasmid pPIC9K and then transformed Pichia pastoris GS115 by electroporation. After colony screening, the bacterium was cultured and the product was purified with affinity chromatography. The affinity of the recombinant human RI with double site mutation was examined for RNase A and its anti-oxidative effect. Results indicated that there were not many changes in the affinity for RNase A detected when compared with the wild type of RI. But the capacity of anti-oxidative effect increased by 7∼9 times. The enhancement in anti-oxidative effect might be attributed to preventing the formation of disulfide bond between Cys328 and Cys329 and the three dimensional structure of RI was thereby maintained. __________ Translated from HEREDITAS, 2005, 27(2) [译自: 遗传,2005,27(2)]     

Evaluation of the role of electrostatic residues in human epidermal growth factor by site-directed mutagenesis and chemical modification.

Four residues in the carboxy-terminal domain of human epidermal growth factor (hEGF), glutamate 40, glutamine 43, arginine 45, and aspartate 46 were targeted for site-directed mutagenesis to evaluate their potential role in epidermal growth factor (EGF) receptor-ligand interaction. One or more mutations were generated at each of these sites and the altered recombinant hEGF gene products were purified and evaluated by radioreceptor competition binding assay. Charge-conservative replacement of glutamate 40 with aspartate resulted in a decrease in receptor binding affinity to 30% relative to wild-type hEGF. On the other hand, removal of the electrostatic charge by substitution of glutamate 40 with glutamine or alanine resulted in only a slightly greater decrease in receptor binding to 25% relative receptor affinity. The introduction of a positive charge upon substitution of glutamine 43 with lysine had no effect on receptor binding. The substitution of arginine 45 with lysine also showed no effect on receptor binding, unlike the absolute requirement observed for the arginine side-chain at position 41 [Engler DA, Campion SR, Hauser MR, Cook JS, Niyogi, SK: J Biol Chem 267:2274-2281, 1992]. Subsequent elimination of the positive charge of lysine 45 by reaction with potassium cyanate showed that the electrostatic property of the residue at this site, as well as that at lysine 28 and lysine 48, was not required for receptor-ligand association.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degeneration and domestication of a selfish gene in yeast: molecular evolution versus site-directed mutagenesis

VDE is a homing endonuclease gene in yeasts with an unusual evolutionary history including horizontal transmission, degeneration, and domestication into the mating-type switching locus HO. We investigate here the effects of these features on its molecular evolution. In addition, we correlate rates of evolution with results from site-directed mutagenesis studies. Functional elements have lower rates of evolution than degenerate ones and higher conservation at functionally important sites. However, functionally important and unimportant sites are equally likely to have been involved in the evolution of new function during the domestication of VDE into HO. The domestication event also indicates that VDE has been lost in some species and that VDE has been present in yeasts for more than 50 Myr.