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KMID : 0364219960390030248
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Korean Journal of Zoology
1996 Volume.39 No. 3 p.248 ~ p.256
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Molecular Cloning and Expression of Human Poly (ADP-ribose) Synthetase cDNA in E. Coli
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Lee Sung-Yong
Kim Wan-Ju
Lee Tae-Sung
Park Sang-Dai
Lee Jung-Sup
Park Jong-Kun
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Abstract
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The present study was performed to clone and express human poly (ADP-ribose) synthetase (PARS) cDNA in E coli. For these purposes, the CDNA for human poly (ADP-ribose) synthetase, encoding the entire protein, was cloned into pGEM-7Zf(+). The resulting recombinant plasmid pPARS6.1 was restriction enzyme mapped and its identity was confirmed by Southern blot analysis. The pPARS6. 1 contained full-length CDNA of human PARS and the nudeotide sequences were identical with those reported previously. The recombinant protein which migrated as a unique 120 kDa band on 10% SDS-polyacrylamide gels, was identified as PARS by Southwestern blots using nick-translated DNA probes and by activity gels and activity blots using 32 P-NAD as a substrate for poly (ADP-ribose) synthetase (PARS). The signals corresponding to 120 and 98 kDa proteins were obtained following IPTG (0.4 mM) induction of the PARS cDNA cloned into Xba I-digested pGEM-7Zf(+) vector. Nonspecific signals corresponding to 45 and 38 kDa proteins were also shown in both IPTG-induced and noninduced cells. The nonspecific proteins may be products of incomplete translation or proteolytic products of intact PARS.
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KEYWORD
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Human Poly(ADP-ribose)synthetase cDNA, Expression, E. coli, Activity Bloty, Southwestern Blot
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