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Title 

Matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF)-based cloning of enolase, ENO1, from Cryphonectria parasitica

Authors 

M J KimH J ChungS M ParkSung Goo ParkD K ChungM S YangD H Kim

Publisher 

The Korean Society for Applied Microbiology

Issue Date 

2004

Citation 

Journal of Microbiology and Biotechnology, vol. 14, no. 3, pp. 620-627

Keywords 

2-D PAGEC. parasiticaenolaseESI-MSMALDI-TOFcryphonectria parasiticamatrix assisted laser desorption ionization time of flight mass spectrometrymolecular cloningcryphonectria

Abstract 

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 a 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 λ 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. coli-expressed 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.

ISSN 

1017-7825

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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