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KMID : 0545120040140030620
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Journal of Microbiology and Biotechnology
2004 Volume.14 No. 3 p.620 ~ p.627
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Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF)-Based Cloning of Enolase, ENO1, from Cryphonectria parasitica
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Kim MJ
Chung HJ/Park SM/Park SG/Chung DK/Yang MS/Kim DH
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Abstract
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On the foundation of a database of genome sequences and protein analyses the ability to clone a gene based on a peptide analysis is becoming more feasible and effective for identifying a specific gene and its protein product of interest. As such the current study conducted protein analysis using 2-D PAGE followed by MALDI-TOF and ESI-MS to identify a highly expressed gene product of C. parasitica. A distinctive and highly expressed protein spot with a molecular size of 47.2 kDa was randomly selected and MALDI-TOF MS analysis was conducted. A homology search indicated that the protein appeared to be a fungal enolase (eno1). Meanwhile multiple alignments of fungal enolases revealed a conserved amino acid sequence from which degenerated primers were designed. A screening of the genomic l library of C. parasitica using the PCR amplicon as a probe was conducted to obtain the full-length gene while RT-PCR was performed for the cDNA. The E. coliexpressed eno1 exhibited enolase enzymatic activity indicating that the cloned gene encoded the C. parasitica enolase. Moreover ESI-MS of two of the separated peptides resolved from the protein spot on 2-D PAGE revealed sequences identical to the deduced sequences suggesting that the cloned gene indeed encoded the resolved protein spot. Northern blot analysis indicated a consistent accumulation of an eno1 transcript during the cultivation.
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