RRC ID 35736
Author Hoseki J, Yano T, Koyama Y, Kuramitsu S, Kagamiyama H.
Title Directed evolution of thermostable kanamycin-resistance gene: a convenient selection marker for Thermus thermophilus.
Journal J Biochem
Abstract The whole-genome sequencing of an extreme thermophile, Thermus thermophilus, is now in progress. Like other genome projects, major concern is shifting from the sequence itself to post-sequencing research such as functional or structural genomics. Under such circumstances, the demand for convenient genetic-engineering tools is increasing. In this study we have increased the thermostability of a kanamycin-resistance gene product using strategies based on directed evolution in T. thermophilus to the upper limit of its growth temperature. The most thermostable mutant has 19 amino-acid substitutions, whereby the thermostability is increased by 20 degrees C, but the enzymatic activity is not significantly changed. Most of the mutated residues are located on the surface of the protein molecule, and, interestingly, five of the 19 substitutions are those to proline residues. The evolved kanamycin-resistance gene products could be used as selection markers at the optimum growth temperature of T. thermophilus. The development of such a convenient genetic-engineering tool would facilitate post-sequencing research on T. thermophilus.
Volume 126(5)
Pages 951-6
Published 1999-11-1
DOI 10.1093/oxfordjournals.jbchem.a022539
PMID 10544290
MeSH Amino Acid Substitution Base Sequence DNA Primers / genetics Directed Molecular Evolution* Enzyme Stability Genes, Bacterial* Genetic Markers Kanamycin Resistance / genetics* Kinetics Models, Molecular Mutation Nucleotidyltransferases / chemistry Nucleotidyltransferases / genetics Nucleotidyltransferases / metabolism Protein Conformation Temperature Thermus thermophilus / enzymology Thermus thermophilus / genetics* Thermus thermophilus / growth & development
IF 2.476
Times Cited 124
DNA material pUC18-HTK (with promoter) (RDB03436)