New insights into the QuikChangeTM process guide the use of Phusion DNA polymerase for site-directed mutagenesis

Xia, Yongzhen; Chu, Wenqiao; Qi, Qingsheng; Xun, Luying

doi:10.1093/nar/gku1189

Cited by 136 publications

(118 citation statements)

References 23 publications

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“…Mutagenesis primers were synthesized by Integrated DNA Technologies, Inc. Mutagenesis of Trp 11 , Gln 58 , Arg 68 , Glu 70 , Tyr 71 , Pro 291 and Gln 294 to Ala was performed according to a modified site-directed mutagenesis method (Xia et al, 2015). Plasmids were purified using QIAprep Spin Miniprep kits (Qiagen).…”

Section: Mutagenesis Of Emoamentioning

confidence: 99%

Structural and biochemical characterization of EDTA monooxygenase and its physical interaction with a partner flavin reductase

Jun

Lewis

Youn

et al. 2016

Molecular Microbiology

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Summary Ethylenediaminetetraacetate (EDTA) is currently the most abundant organic pollutant due to its recalcitrance and extensive use. Only a few bacteria can degrade it, using EDTA monooxygenase (EmoA) to initiate the degradation. EmoA is an FMNH2-dependent monooxygenase that requires an NADH:FMN oxidoreductase (EmoB) to provide FMNH2 as a cosubstrate. Although EmoA has been identified from Chelativorans (ex. Mesorhizobium) sp. BNC1, its catalytic mechanism is unknown. Crystal structures of EmoA revealed a domain-like insertion into a TIM-barrel, which might serve as a flexible lid for the active site. Docking of MgEDTA2− into EmoA identified an intricate hydrogen bond network connected to Tyr71, which should potentially lower its pKa. Tyr71, along with nearby Glu70 and a peroxy flavin, facilitates a keto-enol transition of the leaving acetyl group of EDTA. Further, for the first time, the physical interaction between EmoA and EmoB was observed by ITC, molecular docking and enzyme kinetic assay, which enhanced both EmoA and EmoB activities probably through coupled channeling of FMNH2.

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Section: Mutagenesis Of Emoamentioning

confidence: 99%

Structural and biochemical characterization of EDTA monooxygenase and its physical interaction with a partner flavin reductase

Jun

Lewis

Youn

et al. 2016

Molecular Microbiology

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“…This method involves amplifying the entire plasmid using primers that contain the desired changes, its advantages include time-saving, simplicity and the need for relatively small amounts of template plasmid DNA [19,20]. The new method used site-directed mutagenesis PCR to modify the MCS region of pGADT7, a common prey vector in yeast two-hybrid systems, by replacing its 29 bp Eco RI downstream sequence with the 15 bp Eco RI downstream sequence from the pGBKT7 vector.A new prey vector, pGADT7-In, was then generated, which has the same end sequence as pGBKT7 when linearized by digestion with the restriction enzyme Eco RI.…”

Section: Resultsmentioning

confidence: 99%

A novel, easy and rapid method for constructing yeast two-hybrid vectors using In-Fusion technology

Liao²,

Zhang

et al. 2018

BioTechniques

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Yeast two-hybrid systems are powerful tools for analyzing interactions between proteins. Vector construction is an essential step in yeast two-hybrid experiments, which require bait and prey plasmids. In this study, we modified the multiple cloning site sequence of the yeast plasmid pGADT7 by site-directed mutagenesis PCR to generate the pGADT7-In vector, which resulted in an easy and rapid method for constructing yeast two-hybrid vectors using the In-Fusion cloning technique. This method has three key advantages: only one pair of primers and one round of PCR are needed to generate bait and prey plasmids for each gene, it is restriction endonuclease- and ligase-independent, and it is fast and easily performed.

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“…The amplified fragment was ligated with the vector pET22b to construct the recombinant plasmid pET22b-est22. Site-directed mutations in Est22 were introduced by using the QuikChange mutagenesis method (32), with partially overlapping primers containing mutations (Table 4) and with plasmid pET22b-est22 as the template. Wild-type Est22 and its mutants were expressed in E. coli BL21(DE3) cells for 40 h at 15°C, with induction with 0.5 mM isopropyl-␤-D-thiogalactopyranoside (IPTG).…”

Section: Methodsmentioning

confidence: 99%

A Novel Subfamily Esterase with a Homoserine Transacetylase-like Fold but No Transferase Activity

Yao

Wang

et al. 2017

Appl Environ Microbiol

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Microbial esterases play important roles in deep-sea organic carbon degradation and cycling. Although they have similar catalytic triads and oxyanion holes, esterases are hydrolases and homoserine transacetylases (HTAs) are transferases. Because two HTA homologs were identified as acetyl esterases, the HTA family was recently divided into the bona fide acetyltransferase subfamily and the acetyl esterase subfamily. Here, we identified and characterized a novel HTA-like esterase, Est22, from a deep-sea sedimentary metagenomic library. Est22 could efficiently hydrolyze esters with acyl lengths of up to six carbon atoms but had no transacetylase activity, which is different from HTAs and HTA-like acetyl esterases. Phylogenetic analysis also showed that Est22 and its homologs form a separate branch of the HTA family. We solved the structures of Est22 and its L374D mutant and modeled the structure of the L374D mutant with p-nitrophenyl butyrate. Based on structural, mutational, and biochemical analyses, Phe 71 and Met 176 in the oxyanion hole and Arg 294 were revealed to be the key substrate-binding residues. A detailed structural comparison indicated that differences in their catalytic tunnels lead to the different substrate specificities of Est22 and the other two HTA subfamilies. Biochemical and sequence analyses suggested that Est22 homologs may have the same substrate recognition and catalysis mechanisms as Est22. Due to the significant differences in sequences, structures, and substrate specificities between Est22 (and its homologs) and the other two HTA subfamilies, we suggest that Est22 and its homologs represent a new subfamily in the HTA family.IMPORTANCE Microbial esterases play important roles in the turnover of organic carbon in the deep sea. Esterases and HTAs represent two groups of ␣/␤ hydrolases. Esterases catalyze the hydrolysis of simple esters and are widely used in the pharmaceutical and agrochemical industries, while HTAs catalyze the transfer of an acetyl group from acetyl-coenzyme A (CoA) to homoserine and are essential for microbial growth. Here, we report on a novel HTA-like esterase, Est22, from a deep-sea sediment. Because of the significant differences in sequences, structures, and substrate specificities of HTAs and HTA-like acetyl esterases, Est22 and its homologs represent a new subfamily in the HTA family. This study offers new knowledge regarding marine esterases.

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New insights into the QuikChangeTM process guide the use of Phusion DNA polymerase for site-directed mutagenesis

Cited by 136 publications

References 23 publications

Structural and biochemical characterization of EDTA monooxygenase and its physical interaction with a partner flavin reductase

Structural and biochemical characterization of EDTA monooxygenase and its physical interaction with a partner flavin reductase

A novel, easy and rapid method for constructing yeast two-hybrid vectors using In-Fusion technology

A Novel Subfamily Esterase with a Homoserine Transacetylase-like Fold but No Transferase Activity

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