Characterization of heat-labile uracil-DNA glycosylase from Psychrobacter sp. HJ147 and its application to the polymerase chain reaction.
The gene encoding Psp HJ147 UDG (Psychrobacter sp. HJ147 uracil-DNA glycosylase) was cloned and sequenced. The gene consists of 735 bp for coding a protein with 244 amino acid residues. The deduced amino acid sequence of Psp HJ147 UDG showed a high similarity to that of Psychrobacter articus, Psychrobacter cryohalolentis K5 and Psychrobacter sp. PRwf-1. The PCR-amplified Psp HJ147 UDG gene was expressed under the control of the T7lac promoter on pTYB1 in Escherichia coli BL21(DE3). The expressed enzyme was purified with IMPACT-CN (intein-mediated purification with an affinity chitin-binding tag) system. The optimum pH and temperature of the purified enzyme were 7.0-7.5 and 20-25 degrees C respectively. The optimum NaCl and KCl concentrations for the activity of the purified enzyme ranged from 50 to75 mM. The half-life of the enzyme at 50 degrees C was approx. 45 s. These heat-labile characteristics enabled Psp HJ147 UDG to control carry-over contamination in direct PCR without loss of the PCR product. Psp HJ147 UDG's contaminant control in both direct PCR and indirect PCR exhibited superiority over the UDG of the marine psychrophilic bacterium strain BMTU 3346 and that of E. coli.
Lee
Mi Sun
MS
Department of Genetic Engineering, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746, Republic of Korea.
Kim
Gun A
GA
Seo
Moo Seok
MS
Lee
Jung-Hyun
JH
Kwon
Suk-Tae
ST
D000595:Amino Acid Sequence; D055162:Biocatalysis; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D006358:Hot Temperature; D016133:Polymerase Chain Reaction / Q000379:methods*; D044096:Psychrobacter / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D051981:Uracil-DNA Glycosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D051981:Uracil-DNA Glycosidase