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Ultrafast solvation dynamics at internal sites of staphylococcal nuclease investigated by site-directed mutagenesis

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Chinese Physics B 2015年第1期24卷 81-88页

作者：高光宇李渝王伟王树峰 Dongping Zhong 龚旗煌 Institute of Modern Optics & State Key Laboratory for Artificial Microstructure and Mesoscopic Physics School of PhysicsPeking University Departments of Physics Chemistry and BiochemistryPrograms of BiophysicsChemical Physics and BiochemistryThe Ohio State UniversityColumbusOH 43210USA Collaborative Innovation Center of Quantum Matter

Internal solvation of protein was studied by site-directed mutagenesis, with which an intrinsically fluorescent probe,tryptophan, is inserted into the desired position inside a protein molecule for ultrafast spectroscopic study. Here we review this unique method for protein dynamics research. We first introduce the frontiers of protein solvation, site-directed mutagenesis, protein stability and characteristics, and the spectroscopic methods. Then we present time-resolved spectroscopic dynamics of solvation dynamics inside cavities of active sites. The studies are carried out on a globular protein, staphylococcal nuclease. The solvation at sites inside the protein molecule＇s cavities clearly reveals characteristics of the local environment. These solvation behaviors are directly correlated to enzyme activity.

关键词： ultrafast spectroscopy protein dynamics staphylococcal nuclease(SNase) site-directed mutagenesis

Increased L-arginine Production by site-directed mutagenesis of N-acetyl-L-glutamate Kinase and pro B Gene Deletion in Corynebacterium crenatum

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Biomedical and Environmental Sciences 2015年第12期28卷 864-874页

作者： ZHANG Bin WAN Fang QIU Yu Lou CHEN Xue Lan TANG Li CHEN Jin Cong XIONG Yong Hua Key Laboratory of Functional Small Organic Molecule Ministry of Educationand School of Life ScienceJiangxi Normal University State Key Laboratory of Food Science and Technology Nanchang University

Objective In Corynebacterium crenatum,the adjacent D311 and D312 of N-acetyl-L-glutamate kinase(NAGK),as a key rate-limiting enzyme of L-arginine biosynthesis under substrate regulatory control by arginine,were initially replaced with two arginine residues to investigate the L-arginine feedback inhibition for *** NAGK enzyme expression was evaluated using a plasmid-based *** recombination was employed to eliminate the pro *** The IC50 and enzyme activity of NAGK M4,in which the D311 R and D312 R amino acid substitutions were combined with the previously reported E19 R and H26 E substitutions,were 3.7-fold and 14.6% higher,respectively,than those of the wild-type *** M4 was successfully introduced into the *** MT genome without any genetic markers;the L-arginine yield of *** MT-M4 was 26.2% higher than that of *** *** further improve upon the L-arginine yield,we constructed the mutant *** MT-M4 ?pro *** optimum concentration of L-proline was also investigated in order to determine its contribution to L-arginine *** L-proline was added to the medium at 10 mmol/L,the L-arginine yield reached 16.5 g/L after 108 h of shake-flask fermentation,approximately 70.1% higher than the yield attained using *** *** Feedback inhibition of L-arginine on NAGK in *** is clearly alleviated by the M4 mutation of NAGK,and deletion of the pro B in *** from MT to M4 results in a significant increase in arginine production.

关键词： Corynebacterium crenatum N-acetyl-L-glutamate kinase site-directed mutagenesis L-arginine proB

Activity after site-directed mutagenesis of CD59 on Complement-Mediated Cytolysis

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Cellular & Molecular Immunology 2008年第2期5卷 141-146页

作者： Xinhong Zhu Meihua Gao Shurong Ren Qiubo Wang Cunzhi Lin Department of Immunology Medical College of Qingdao University Qingdao Shandong China Clinical Immune Institution Qingdao Chest Hospital 896 Chongqing Road Qingdao 266043 China

CD59 may inhibit the cytolytic activity of complement by binding to C8/C9 and protect host cell membranes against homologous membrane attack complex （MAC）. However, CD59 is widely overexpressed on tumor cells, which has been implicated in tumorigenesis. The active site of CD59 relative to MAC is still confused. As reported the MAC binding site is located in the vicinity of a hydrophobic groove on the membrane distal face of the protein centered around residue W40. Here two site-directed mutagenesis were performed by overlapping extension PCR to delete residue W40 site （Mutant 1, M1） or to change C39W40K41 to W39W40W41 （Mutant 2, M2）. Then we constructed mutant CD59 eukaryotic expression system and investigated their biological function on CI-IO cells compared with wild-type CD59. Stable populations of CHO cells expressing recombinant proteins were screened by immunotechnique. After 30 passages culturing, proteins could be tested. Dye release assays suggest that M1CD59 loses the activity against complement, while M2CD59 increases the anti-complement activity slightly. Results indicate that W40 of human CD59 is important to its activity, and prohibition of this site may be a potential way to increase complement activity and to treat tumors. Cellular ＆ Molecular Immunology.

关键词： CD59 complement active site site-directed mutagenesis overlap.extension PCR

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

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Frontiers in Biology 2006年第2期1卷 99-103页

作者： Wu Yu Cui Xiuyun Wang Jihong Zhao Peng Xu Yuefei Zhao Baochang College of Live Sciences Liaoning Normal UniversityDalian 116029China Department of Biochemistry and Molecular Biology Dalian Medical UniversityDalian 116027China

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

关键词： ribonuclease inhibitor site-directed mutagenesis cysteine Pichia pastoris reactive oxygen species

Improvement of the Optimum Temperature of Penicillium expansum Lipase by site-directed mutagenesis

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Frontiers in Biology 2006年第1期1卷 1-4页

作者： Chen Guoren Lin Lin Bioengineering College Fujian Normal UniversityFuzhou 350007China

In order to improve the optimum temperature of lipases,the Penicillum expansum lipase(PEL)gene was mutated by site-directed mutagenesis using overlap extension PCR *** recombinant plasmid containing mutant E83V pPIC3.5K-lip-E83V was expressed in Pichia pastoris *** experiments of the mutant PEL-E83V-GS and the wild-type PEL-GS showed that the optimum temperature(45℃)of the mutant was 5℃ higher than that of the wild *** thermostability of the mutant was similar to that of the wild *** enzymatic activity of the mutant was 188 U/ml at 37℃,which was 80%that of the wild type in the same *** interaction may be enhanced in the surface region by the hydrophilic amino acid Glu substituted with the hydrophobic amino acid Val,and may be responsible for the improvement of the optimum temperature.

关键词： Penicillum expansum lipase site-directed mutagenesis optimum temperature

Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2016年第2期17卷 83-99页

作者： Jian-zhong XU Wei-guo ZHANG The Key Laboratory of Industrial Biotechnology Ministry of Education School of Biotechnology Jiangnan University

With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators.

关键词： Escherichia coli Corynebacterium glutamicum DNA manipulation site-directed mutagenesis Gene inactivation Gene over-expression

Activation of anthrachamycin biosynthesis in Streptomyces chattanoogensis L10 by site-directed mutagenesis of rpoB

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2019年第12期20卷 983-994页

作者： Zi-yue LI Qing-ting BU Jue WANG Yu LIU Xin-ai CHEN Xu-ming MAO Yong-Quan LI Institute of Pharmaceutical Biotechnology&First Affiliated Hospital School of MedicineZhejia ng UniversityHangzhou 310058China Zhejiang Provincial Key Laboratory for Microbial Biochemistry and Metabolic Engineering Hangzhou 310058China College of Life Sciences Zhejiang UniversityHangzhou 310058China

Genome sequencing projects revealed massive cryptic gene clusters encoding the undiscovered secondary metabolites in Streptomyces. To investigate the metabolic products of silent gene clusters in Streptomyces chattanoogensis L10(CGMCC 2644), we used site-directed mutagenesis to generate ten mutants with point mutations in the highly conserved region of rpsL(encoding the ribosomal protein S12) or rpoB(encoding the RNA polymerase β-subunit). Among them, L10/RpoB(H437 Y) accumulated a dark pigment on a yeast extract-malt extract-glucose(YMG) plate. This was absent in the wild type. After further investigation, a novel angucycline antibiotic named anthrachamycin was isolated and determined using nuclear magnetic resonance(NMR) spectroscopic techniques. Quantitative real-time polymerase chain reaction(qRT-PCR) analysis and electrophoretic mobility shift assay(EMSA) were performed to investigate the mechanism underlying the activation effect on the anthrachamycin biosynthetic gene cluster. This work indicated that the rpoB-specific missense H437 Y mutation had activated anthrachamycin biosynthesis in S. chattanoogensis L10. This may be helpful in the investigation of the pleiotropic regulation system in Streptomyces.

关键词： Streptomyces Cryptic gene cluster site-directed mutagenesis Secondary metabolism

Mechanism of multi-point site-directed mutagenesis

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Progress in Natural Science:Materials International 1997年第3期 48-55页

作者：谢毅肖谷田林卿王顺德叶韫辉吴小舟

Subtilisin E gene was mutagemzed in vitro as a model in one-round mutagenesis with multiple chemical-synthesized mutagenic oligonucleotides. The rationale of the new method is suggested The elements that influence the mutant efficiency such as the type, amount, distribution and mutual location of primers, and the primer to template molar ratio are discussed

关键词： site-directed mutagenesis multi-point site-specific mutagenesis protein engineering subtilisin E.

site-directed mutagenesis of Streptomyces avermitilis aveD Gene

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Agricultural Science & Technology 2011年第10期12卷 1424-1426页

作者：汤晖张利平河北大学生命科学学院河北省微生物多样性研究与应用实验室河北保定071002

[Objective] The aim of this study was to produce Streptomyces avermitilis strain with site-directed mutagenesis in aveD gene,and so as to provide theoretical basis for genetic breeding of ***.[Method] PCR-driven overlap extension was conducted for the site-directed mutagenesis in aveD gene;the mutated aveD gene then was used to construct vector pDC3（pKC1139∷aveD） via molecular manipulations like in vitro enzyme digestion and ligation;the vector pDC3（pKC1139∷aveD） was then introduced to aveD deletion mutant 489 of avermectin-producing strain *** 76-9.[Result] Mutant strain 536 of site-directed mutagenesis of *** 76-9 was obtained by homologous *** sequencing results show that the sixty-ninth base C in aveD-coding region of mutant 536 was substituted by T,and the corresponding amino acid Thr was mutated to be Ile.[Conclusion] This study laid basis for the development of strains specifically producing avermectin B.

关键词： PCR-driven overlap extension aveD gene site-directed mutagenesis