Substitution of Ile(707) for Leu in Klentaq DNA polymerase reduces the amplification capacity of the enzyme.
The high yield and specificity of PCR amplifications are affected by DNA polymerase activity at room temperature. One way of preventing this unwanted activity is by genetic modifications of the DNA polymerase. For Taq DNA polymerase, mutations in the gene (Glu626Lys, Trp706Arg, Ile707Leu and Glu708Asp), when introduced individually or in certain combinations, were found to contribute to a significant decrease of the enzyme activity at room temperature. The aim of this study was to evaluate the usefulness of the Ile707Leu cold-sensitive mutation in the N-terminal deletional variant of Taq DNA polymerase in PCR reaction. The Ile(707) to Leu substitution was introduced to Klentaq278 by site-directed mutagenesis. Normal and mutant DNA polymerases were expressed under a tac promoter and purified to homogeneity. The mutant polymerase showed reduced polymerase activity at room temperature by up to 12 times and no significant change in thermostability, compared to Klentaq278 DNA polymerase. The major effect of the amino acid substitution was the reduction of the amplification capacity of the polymerase. Mutant polymerase could not amplify fragments over 1 kb. In conclusion, the substitution of Ile707Leu in Klentaq278 DNA polymerase reduces the overall processivity of the enzyme and therefore limits the application of this DNA polymerase in PCR.
Davalieva
K
K
Research Centre for Genetic Engineering and Biotechnology, Macedonian Academy of Sciences and Arts, Krste Misirkov 2, 1000 Skopje, R. Macedonia.
Efremov
D G
DG
D019943:Amino Acid Substitution; D007532:Isoleucine / Q000235:genetics*; D007930:Leucine / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D019914:Taq Polymerase / Q000235:genetics* / Q000378:metabolism; D013696:Temperature
D007532:Isoleucine; D019914:Taq Polymerase; D007930:Leucine