PCR-Based Random Mutagenesis Using Manganese and Reduced dNTP Concentration

Lin-Goerke, Juili; Robbins, David J.; Burczak, John D.

doi:10.2144/97233bm12

Cited by 107 publications

(76 citation statements)

References 8 publications

(13 reference statements)

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“…The mutations were distributed evenly inside the PCR amplified region. Interestingly, comparison of our data with the ones given by Lin-Goerke et al (10), in which the transversion of GC →CG was only 1.4%, the frequency we achieved with our method (8.3%) is considerably higher. This may be due to the preferential order of dITP for pairing of bp (12).…”

supporting

confidence: 66%

Random Mutagenesis Libraries: Optimization and Simplification by PCR

Petersen

et al. 1999

BioTechniques

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supporting

confidence: 66%

Random Mutagenesis Libraries: Optimization and Simplification by PCR

Petersen

et al. 1999

BioTechniques

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“…For random mutagenesis, the vao gene in pBC11 was amplified using manganese-based error-prone PCR mutagenesis (34,35). 25 l of a PCR mixture containing 10 pmol of primers 5Ј-GTA-AAACGACGGCCAGT-3Ј and 5Ј-CAGGAAACAGCTATGAC-3Ј, 90 ng of template DNA, and 250 or 500 pmol of MnCl 2 was heated for 3 min at 95°C and, after cooling down to 80°C, mixed with 25 l of a mixture containing 0.4 mM each dATP and dGTP, 2 mM each dCTP and dTTP, 2.5 units of Taq DNA polymerase, 20 mM Tris-HCl (pH 8.85), 4 mM MgCl 2 , 50 mM KCl, and 10 mM (NH 4 ) 2 SO 4 in a thin-wall PCR tube.…”

Section: Methodsmentioning

confidence: 99%

Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

Heuvel

Berg

Rovida

et al. 2004

Journal of Biological Chemistry

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The flavoenzyme vanillyl-alcohol oxidase was subjected to random mutagenesis to generate mutants with enhanced reactivity to creosol (2-methoxy-4-methylphenol). The vanillyl-alcohol oxidase-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) is oxidized to the widely used flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde). The first step of this reaction is extremely slow due to the formation of a covalent FAD N-5-creosol adduct. After a single round of error-prone PCR, seven mutants were generated with increased reactivity to creosol. The single-point mutants I238T, F454Y, E502G, and T505S showed an up to 40-fold increase in catalytic efficiency (k cat /K m ) with creosol compared with the wild-type enzyme. This enhanced reactivity was due to a lower stability of the covalent flavin-substrate adduct, thereby promoting vanillin formation. The catalytic efficiencies of the mutants were also enhanced for other ortho-substituted 4-methylphenols, but not for p-cresol (4-methylphenol). The replaced amino acid residues are not located within a distance of direct interaction with the substrate, and the determined three-dimensional structures of the mutant enzymes are highly similar to that of the wild-type enzyme. These results clearly show the importance of remote residues, not readily predicted by rational design, for the substrate specificity of enzymes.The increased use of enzymes and other proteins in the pharmaceutical, chemical, and agricultural industry has generated considerable interest in the design of proteins with new or improved properties. Two different but complementary technologies have been applied to this goal: (i) rational design, which relies on the availability of the three-dimensional structure and knowledge about the relationship between sequence, structure, and mechanism, and (ii) directed evolution methods, which use random mutagenesis of the gene encoding the protein or recombination of gene fragments to create diversity and then experimental screening of the libraries generated for the desired properties. Rational design has been used to elucidate and change enzyme mechanism, substrate and product specificity, enantioselectivity, cofactor specificity, and protein stability (1-3). Directed evolution has been applied to increase catalytic activity; to invert or improve enantioselectivity; and to alter substrate and product specificity, protein stability, pH optimum, and tolerance against organic solvents (1, 4 -7). An obvious advantage of directed evolution methods over sitedirected mutagenesis is that enzymes can be tailored for the production of (intermediate) products without detailed knowledge of protein structure and structure-function relationships.In this study, we engineered the flavoenzyme vanillyl-alcohol oxidase (VAO) 1 by random mutagenesis such that the evolved mutants are capable of producing natural vanillin (4-hydroxy-3-methoxybenzaldehyde) from the precursor creosol (2-methoxy...

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“…We mutagenized Caspase 2 by using Mn 2ϩ -biased PCR as described (22,23) with primers 5Ј-CTCTCA AT TGGCCACCATGGCCGCTGA-CAGGGGACGCAG-3Ј and 5Ј-GCTCCTCGCCCTTGCT-CACCATTGTGGGAGGGTGTCCTGGGAAC-3Ј and Taq polymerase for 40 cycles. We amplified enhanced GFP (EGFP) by using PfuTurbo polymerase with 5Ј-GTTCCCAGGACAC-CCTCCCACAATGGTGAGCAAGGGCGAGGAGC-3Ј and 5Ј-CTCCGTCGACCTCGAGTTACTTGTACAGCTCGTC-CATGC-3Ј.…”

Section: Methodsmentioning

confidence: 99%

Cyclin D3 activates Caspase 2, connecting cell proliferation with cell death

Mendelsohn

Hamer²,

Wang³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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We describe a PCR mutagenesis͞ligation͞two-hybrid͞green fluorescent protein approach that facilitates the isolation of missense mutant proteins defective in interaction with particular partners absent other phenotypes or knowledge of the system. We used this approach to isolate Caspase 2 mutants that did not bind cyclin D3 (noninteractors). Noninteractors were sensitive to apoptosis-dependent proteolysis, but did not potentiate apoptosis. Noninteractors did not block apoptosis caused by wildtype Caspase 2. Our results are consistent with the idea that an interaction with cyclin D3 may stabilize Caspase 2, and suggest that a physical interaction between cyclin D3 and Caspase 2 connects the genetic networks that govern cell-cycle progression with those that govern cell death.

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PCR-Based Random Mutagenesis Using Manganese and Reduced dNTP Concentration

Cited by 107 publications

References 8 publications

Random Mutagenesis Libraries: Optimization and Simplification by PCR

Random Mutagenesis Libraries: Optimization and Simplification by PCR

Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

Cyclin D3 activates Caspase 2, connecting cell proliferation with cell death

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