Molecular cloning, expression, and site-directed mutagenesis of inorganic pyrophosphatase from Thermus thermophilus HB8.
The genomic DNA encoding the inorganic pyrophosphatase from an extremely thermophilic bacterium, Thermus thermophilus HB8 (ATCC27634), was isolated by colony hybridization with a probe designed as a part of gene amplified by the PCR method, which was derived from the partial amino acid sequence of the enzyme. The DNA was cloned into a plasmid vector, pUC118, after digestion with BamHI. The inserted nucleotide fragment was about 1.8 kbp in length and the nucleotide sequence included a 525 bp open reading frame. The deduced amino acid sequence was completely identical with that of the enzyme determined by automated Edman analysis of peptide fragments isolated from digests obtained with Staphylococcus aureus V8 protease and Achromobacter protease I, and also from products obtained on chemical cleavage with cyanogen bromide and 70% formic acid. The subunit of this enzyme is composed of 174 amino acid residues with a calculated molecular weight of 19,084. Then, the gene was overexpressed in Escherichia coli BL21 (DE3) using a plasmid vector, pET15b, system. The recombinant enzyme was fully active, and exhibited higher thermostability than the E. coli enzyme. Amino acid residues located on the surface of the recombinant enzyme were determined by means of limited proteolysis, and the results revealed that the environment of Lys residues is almost the same as the crystal structure reported previously [Teplyakov, A. et al. (1994) Protein Sci. 3, 1098-1107]. Furthermore, the roles of two tryptophan residues were investigated by site-directed mutagenesis, which indicated that they may be responsible for the structural integrity and thermostability.
Satoh
T
T
Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University, Chitosedai, Setagaya-ku, Tokyo, 157-8572, Japan.
Samejima
T
T
Watanabe
M
M
Nogi
S
S
Takahashi
Y
Y
Kaji
H
H
Teplyakov
A
A
Obmolova
G
G
Kuranova
I
I
Ishii
K
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000201:enzymology*; D014364:Tryptophan / Q000737:chemistry
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D014364:Tryptophan; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase