[Expression, purification and enzymatic characterization of adenylate kinase of Thermus thermophilus HB27 in Escherichia coli].
OBJECTIVE
To clone the gene encoding adenylate kinase of Thermus thermophilus HB27, an extremely thermophilic bacterium, express the protein in Escherichia coil and study the enzymatic characterization.


METHODS
The DNA fragment encoding adenylate kinase was obtained by PCR from the total DNA of Thermus thermophilus HB27 and cloned into the vector pET-28a. The recombinant plasmid was identified by PCR, restriction endonuclease digestion and sequence analysis. Enzymatic characterization of the expressed protein was carried out using spectrophotometric assays.


RESULTS
The gene coding for adenylate kinase from Thermus thermophilus HB27 was cloned and the protein was overexpressed in Escherichia coli BL21(DE3). The optimum reactive pH and temperature for the enzyme were 8.5 and 90 degrees celsius;, respectively. The Km of the recombinant adenylate kinase for ADP was 68.6 micromol/L, with an V(max)ADP of 0.294 mmol/(L.min). Under the condition of environmental temperature at 70, 80, 90, or 100 degrees celsius; for 7 h, the recombinant adenylate kinase still retained the enzymatic activity with high thermostability. AP5A, a specific adenylate kinase inhibitor, inhibited the enzymatic activity of the protein by 70% at the concentration of 2.0 mmol/L, with a Ki value of 46.39 micromol/L for ADP.


CONCLUSIONS
The gene coding for adenylate kinase of Thermus thermophilus HB27 has been successfully cloned and expressed in Escherichia coil, which provides the basis for potential use of the highly thermostable recombinant HB27 adenylate kinase.
Tan
Zhi-wen
ZW
Institute of Biochemistry, Zhejiang Sci-Tech University, Hangzhou, 310018, China.
Liu
Jun
J
Zhang
Xue-fang
XF
Meng
Fan-guo
FG
Zhang
Yao-zhoug
YZ
D000263:Adenylate Kinase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D016963:Thermus thermophilus / Q000201:enzymology*
D011994:Recombinant Proteins; D000263:Adenylate Kinase