Asymmetric overlap extension PCR method bypassing intermediate purification and the amplification of wild-type template in site-directed mutagenesis

The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40–45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment.

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Site-Directed Mutagenesis Using Overlap Extension PCR

In Vitro Mutagenesis Protocols ◽

10.1385/0-89603-332-5:177 ◽

2003 ◽

pp. 177-192 ◽

Cited By ~ 30

Author(s):

Ashok Aiyar ◽

Yan Xiang ◽

Jonathan Leis

Keyword(s):

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Overlap Extension Pcr ◽

Overlap Extension

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A rapid and efficient method for site-directed mutagenesis using one- step overlap extension PCR

Nucleic Acids Research ◽

10.1093/nar/25.11.2227 ◽

1997 ◽

Vol 25 (11) ◽

pp. 2227-2228 ◽

Cited By ~ 146

Author(s):

A Urban

Keyword(s):

Efficient Method ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Overlap Extension Pcr ◽

Overlap Extension ◽

One Step

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Enzyme activity enhancement of chondroitinase ABC I from Proteus vulgaris by site-directed mutagenesis

RSC Advances ◽

10.1039/c5ra15220h ◽

2015 ◽

Vol 5 (93) ◽

pp. 76040-76047 ◽

Cited By ~ 11

Author(s):

Zhenya Chen ◽

Ye Li ◽

Yue Feng ◽

Liang Chen ◽

Qipeng Yuan

Keyword(s):

Active Site ◽

Simulation Analysis ◽

Docking Simulation ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Proteus Vulgaris ◽

Molecular Docking Simulation ◽

Activity Enhancement ◽

First Time

Arg660 was found as a new active site and Asn795Ala and Trp818Ala mutants showed higher activities than the wild type based on molecular docking simulation analysis for the first time.

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Mutant Firefly Luciferase Enzymes Resistant to the Inhibition by Sodium Chloride

10.21203/rs.3.rs-243105/v1 ◽

2021 ◽

Author(s):

Satoshi Yawata ◽

Kenichi Noda ◽

Ai Shimomura ◽

Akio Kuroda

Keyword(s):

Sodium Chloride ◽

Double Mutant ◽

Luciferase Activity ◽

Firefly Luciferase ◽

Activity Level ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Original Activity ◽

Double Mutation

Abstract ObjectivesFirefly luciferase, one of the most extensively studied enzymes, has numerous applications. However, luciferase activity is inhibited by sodium chloride. This study aims to expand the applications of firefly luciferase in the presence of sodium chloride.ResultsWe first obtained two mutant luciferase enzymes whose inhibition were alleviated and identified these mutations as Val288Ile and Glu488Val. Under dialysis condition (140 mM sodium chloride), the wild type was inhibited to 44% of its original activity level. In contrast, the single mutants, Val288Ile and Glu488Val, retained 67% and 79% of their original activity, respectively. Next, we introduced Val288Ile and Glu488Val mutations into the wild-type luciferase to create a double mutant using site-directed mutagenesis. Notably, the double mutant retained its activity more than 95% of that in the absence of sodium chloride.ConclusionsThe mutant luciferase, named luciferase CR, was found to retain its activity in various concentrations of sodium chloride. The inhibition of luciferase CR under dialysis condition was more alleviated than either Val288Ile or Glu488Val alone, suggesting that the effect of the double mutation was cumulative. We discussed the effect of mutations on the alleviation of the inhibition by sodium chloride.

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Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis

Biochemical Journal ◽

10.1042/bj2790035 ◽

1991 ◽

Vol 279 (1) ◽

pp. 35-41 ◽

Cited By ~ 86

Author(s):

R Chambert ◽

M F Petit-Glatron

Keyword(s):

Bacillus Subtilis ◽

Intermediate Formation ◽

Site Directed Mutagenesis ◽

Single Amino Acid ◽

Chain Elongation ◽

Directed Mutagenesis ◽

Water Mixture ◽

Wild Type ◽

Wild Type Enzyme ◽

Sucrose Hydrolysis

The levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) structural gene from a Bacillus subtilis mutant strain displaying a low polymerase activity was sequenced. Only one missense mutation changing Arg331 to His was responsible for this modified catalytic property. From this allele we created new mutations by directed mutagenesis, which modified the charge and polarity of site 331. Examination of the kinetics of the purified levansucrase variants revealed that transfructosylation activities are affected differently by the substitution chosen. His331→Arg completely restored the properties of the wild-type enzyme. The most striking feature of the other variants, namely Lys331, Ser331 and Leu331, was that they lost the ability of the wild-type enzyme to synthesize levan from sucrose alone. They were only capable of catalysing the first step of levan chain elongation, which is the formation of the trisaccharide ketose. The variant His331→Lys presented a higher kcat. for sucrose hydrolysis than the wild-type, and only this hydrolase activity was preserved in a solvent/water mixture in which the wild-type acted as a true polymerase. The two other substitutions reduced the efficiency of transfructosylation activities of the enzyme via the decrease of the rate of fructosyl-enzyme intermediate formation. For all variants, the sucrose affinity was slightly affected. This strong modulation of the enzyme specificities from a single amino acid substitution led us to postulate the hypothesis that bacterial levansucrases and plant fructosyltransferases involved in fructan synthesis may possess a common ancestral form.

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ROLE OF PROTEOLYSIS AT ARGININE-275 OF TISSUE PLASMINOGEN ACTIVATOR (t-PA) AS ASSESSED BY SITE-DIRECTED MUTAGENESIS

10.1055/s-0038-1643843 ◽

1987 ◽

Author(s):

G A Vehar ◽

K M Tate ◽

D L Higgins ◽

W E Holmes ◽

H L Heyneker

Keyword(s):

Cho Cells ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Single Chain ◽

Serine Protease Family ◽

The One ◽

Chain Form

The significance of the cleavage at arginine-275 of human t-PA has been the subject of debate. It has been reported, as expected for a member of the serine protease family, that the single chain form is a zymogen and that generation of catalytic activity is dependent upon cleavage at arginine-275. Other groups, in contrast, have found considerable enzyme activity associated with the one-chain form of t-PA. To clarify the functional significance of this proteolysis and circumvent cleavage of one-chain t-PA by itself or plasmin, site-directed mutagenesis was employed to change the codon of arginine-275 to specify a glutamic acid. The resulting plasmid was used to transfect CHO cells. The single chain mutant [Glu-275 t-PA] was expressed in CHO cells and the protein purified by conventional techniques. The mutant enzyme could be converted to the two-chain form by V8 protease, but not by plasmin. Glu-275 t-PA was 8 times less active in the cleavage of a tripeptide substrate and 20-50 times less active in the activation of plasminogen in the absence of firbrin(ogen) than its two-chain form. In the presence of fibrin(ogen), in contrast, the one and two-chain forms of Glu-275 t-PA were equal in their ability to activate plasminogen in the presence of fibrin(ogen). The activity in these assays was equal to the activity of wild type t-PA. In addition, it was observed that fibrin bound considerably more of the one-chain form of t-PA than the two chain forms of t-PA and the Glu-275 mutant. The one and two-chain forms of the wild type and mutated t-PA were found to slowly form complexes with plasma protease inhibitors in vitro, although the one-chain forms were less reactive with alpha-2-macroglobulin. It can be concluded that the one-chain form of t-PA appears to be fully functional under physiologic conditions and has an increased affinity for fibrin compared to two-chain t-PA.

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Implications of Stisa2 catalytic residue restoration through site directed mutagenesis

Turkish Journal of Biochemistry ◽

10.1515/tjb-2016-0169 ◽

2016 ◽

Vol 42 (2) ◽

Author(s):

Hasnain Hussain ◽

Nikson Fatt Ming Chong

Keyword(s):

Catalytic Activity ◽

Catalytic Triad ◽

Site Directed Mutagenesis ◽

Amino Acid Residues ◽

Qualitative And Quantitative Analysis ◽

Conserved Residues ◽

Overlap Extension Pcr ◽

Overlap Extension ◽

Catalytic Network ◽

And Function

AbstractObjective:Restoration of catalytic activity of Isa2 fromMethods:The six conserved amino acid residues absent in the Stisa2 gene were restored by mutation using the overlap extension PCR and the asymmetrical overlap extension PCR methods. Next, mutant Stisa2 with restored catalytic residues was expressed inResults:Both qualitative and quantitative analysis showed that the restoration of the conserved residues in the catalytic site did not restore starch debranching activity. Molecular modeling showed greater than expected distances between the catalytic triad in mutant Stisa2. These additional distances are likely to prevent hydrogen bonding which stabilizes the reaction intermediate, and are critical for catalytic activity.Conclusions:These results suggest that during evolution, mutations in other highly conserved regions have caused significant changes to the structure and function of the catalytic network. Catalytically inactive Isa2, which is conserved in starch-producing plants, has evolved important non-catalytic roles such as in substrate binding and in regulating isoamylase activity.

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