Shigenori Emi

Cloning, nucleotide sequence, and expression in Escherichia coli of DNA polymerase gene (polA) from Thermus thermophilus HB8

Abstract A gene coding for a thermostable DNA polymerase I (Tth Pol I) was cloned from Thermus thermophilus HB8, and its nucleotide sequence was identified. The Tth Pol I gene ( poIA ) has an open reading frame of 2505 base pairs available to encode a peptide of 834 amino acids. Another incomplete open reading frame was also found upstream from the poA gene. In the deduced amino acid sequence of Tth Pol I, which shows 87% similarity to that of Thermus aquaticus DNA polymerase, there is the leucine zipper structure from Leu458 to Leu486. The Tth Pol I gene was subcloned in a high-expression vector. Escherichia coli cells harboring the hybrid plasmid produced about 100,000 units of thermostable DNA polymerase in a 200-ml culture.

Determination of α-amylase using a new blocked substrate (3-ketobutylidene β-2-chloro-4-nitrophenyl-maltopentaoside)

Abstract A new substrate, 3-ketobutylidene β-2-chloro-4-nitrophenylmaltopentaoside (3KB-CNPG5), was used for the determination of α-amylase (EC 3.2.1.1) in serum and urine. Under this α-amylase assay condition, 3KB-CNPG5 is resistant to glucoamylase and α-glucosidase, which are auxiliary enzymes, because the 4- and 6-positions of the non-reducing-end glucose residue are modified by the 3-ketobutylidene group. The assay using 3KB-CNPG5 for α-amylase activity is a highly sensitive method that uses 2-chloro-4-nitrophenol (CNP) as an aglycone, and is a stable method for determination of α-amylase activity in biological fluids.

Construction and characterization of N-terminally truncated DNA polymerase from Thermus thermophilus

Abstract Various plasmids harboring the truncated DNA polymerase gene ( polA ) from Thermus thermophilus HB8 ( Tth polymerase) were constructed. The most thermostable Tth Δ NF 2 polymerase [the gene product of polA Δ NF 2, which lacked a 751-bp region (region flanked by initiation codon and Fsp I site in the polA gene)] was selected, and purified from the recombinant Escherichia coli . SDS polyacrylamide gel electrophoresis revealed that the molecular weight of the Tth Δ NF 2 polymerase is 58–61 kDa, which is approximately 30 kDa smaller than that of the wild-type enzyme. The specific activity of the 5′-to-3′ polymerization of the Tth Δ NF 2 polymerase was 63% of that of the Tth polymerase. However, no 5′-to-3′ exonuclease activity was detected in this mutant enzyme (less than 1% of the specific activity of wild-type enzyme). The activities of the wild-type and mutant enzymes were maximal at 75°C. Approximately 50% of the enzyme activity was retained even after heat treatment of the Tth Δ NF 2 polymerase at 70°C for 2 h, but the thermostability of the mutant enzyme was slightly lower than that of the wild-type enzyme. Both the Tth Δ NF 2 and Tth polymerases were capable of non-templated addition of deoxyribonucleotide to a 3′-hydroxyl group of blunt-ended DNA.