Cloning and thermostability of TaqI endonuclease isoschizomers from Thermus species SM32 and Thermus filiformis Tok6A1.
Two TaqI endonuclease (hereafter referred to as TaqI) isoschizomer genes, tsp32IR from Thermus species SM32 of Azores and tfiTok6A1I from T. filiformis Tok6A1 of New Zealand, were cloned in Escherichia coli. The overexpressed enzymes were partly purified and their thermostability was determined. In the medium-salt buffer, Tsp32IR, TfiTok6A1I and one previously cloned TaqI isoschizomer (TthHB8I) were more thermostable than TaqI. Tsp32IR remained partly active up to 90 degreesC in the low-salt buffer. Six amino acid residues that are identical in the three high thermostability isoschizomers (Tsp32IR, TfiTok6A1I and TthHB8I) but differ in TaqI might provide added rigidity for thermostabilization. These include four proline residues located in or near loop regions, and one alanine and one arginine located at helix regions in the predicted TaqI endonuclease secondary structure. The possible role of these residues in thermostabilization was evaluated by mutagenizing the TaqI enzyme. Mutants generated at these six positions were less thermostable than wild-type TaqI. The results suggest that the surrounding sequence or structural context might be as important as the mutation itself.
Cao
W
W
Department of Microbiology, Hearst Microbiology Research Center, Cornell University Medical College and Strang Cancer Prevention Center, New York, NY 10021, USA.
Lu
J
J
Welch
S G
SG
Williams
R A
RA
Barany
F
F
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D016415:Sequence Alignment; D013696:Temperature; D013824:Thermus / Q000201:enzymology* / Q000254:growth & development
D015252:Deoxyribonucleases, Type II Site-Specific; C027046:TCGA-specific type II deoxyribonucleases