OBJECTIVETo 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.

METHODSThe 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.

RESULTSThe 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.

CONCLUSIONThe 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.

Adenylate Kinase ; biosynthesis ; genetics ; metabolism ; Amino Acid Sequence ; Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Thermus thermophilus ; enzymology

Ribosomal RNAs are important because they catalyze the synthesis of peptides and proteins. Comparative studies of the secondary structure of 18S rRNA have revealed the basic locations of its many length-conserved and length-variable regions. In recent years, many more sequences of 18S rDNA with unusual lengths have been documented in GenBank. These data make it possible to recognize the diversity of the secondary and tertiary structures of 18S rRNAs and to identify the length-conserved parts of 18S rDNAs. The longest 18S rDNA sequences of almost every known eukaryotic phylum were included in this study. We illustrated the bioinformatics-based structure to show that, the regions that are more length-variable, regions that are less length-variable, the splicing sites for introns, and the sites of A-minor interactions are mostly distributed in different parts of the 18S rRNA. Additionally, this study revealed that some length-variable regions or insertion positions could be quite close to the functional part of the 18S rRNA of Foraminifera organisms. The tertiary structure as well as the secondary structure of 18S rRNA can be more diverse than what was previously supposed. Besides revealing how this interesting gene evolves, it can help to remove ambiguity from the alignment of eukaryotic 18S rDNAs and to improve the performance of 18S rDNA in phylogenetic reconstruction. Six nucleotides shared by Archaea and Eukaryota but rarely by Bacteria are also reported here for the first time, which might further support the supposed origin of eukaryote from archaeans.
Animals ; Base Sequence ; Drosophila melanogaster ; genetics ; Eukaryota ; classification ; Molecular Sequence Data ; Nucleic Acid Conformation ; Phylogeny ; RNA, Ribosomal, 16S ; chemistry ; genetics ; RNA, Ribosomal, 18S ; chemistry ; classification ; genetics ; Sequence Alignment ; Sequence Analysis, RNA ; Thermus thermophilus ; genetics

To obtain thermostable aspartate aminotransferase, the gene aspC from an extremely thermophilic bacterium, Thermus thermophilus HB8 was cloned, and its product was overexpressed in Escherichia coli BL21 (DE3) and Rosetta (DE3). The expression in Rosetta (DP3) was more efficient. The optimum reactive pH was 7, and the recombinant enzyme activity changed little when incubated in the buffer of pH8 - 10 on 37 degrees C for 1 h. The optimum reactive temprature was 75 degrees C, and the recombinant enzyme was more stable on the temperature of 25 - 55 degrees C. The half life of recombinant enzyme on 65 degrees C was 3.5 h, on 75 degrees C was 2.5 h. KmKG was 7.559 mmol/L, VmaxKG was 0.086 mmol/(L x min), KmAsp was 2.031 mmol/L, VmaxAsp was 0.024 mmol/(L x min). Ca2+, Fe3+, Mn2+ inhibited enzyme activity softly.
Aspartate Aminotransferases ; genetics ; isolation & purification ; metabolism ; Bacterial Proteins ; genetics ; isolation & purification ; metabolism ; Biocatalysis ; drug effects ; Electrophoresis, Polyacrylamide Gel ; Enzyme Stability ; drug effects ; Escherichia coli ; genetics ; Gene Expression Regulation, Bacterial ; Gene Expression Regulation, Enzymologic ; Hydrogen-Ion Concentration ; Kinetics ; Metals ; pharmacology ; Recombinant Proteins ; isolation & purification ; metabolism ; Temperature ; Thermus thermophilus ; enzymology ; genetics