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Cloning, sequencing, and expression of the gene encoding enolase from Cunninghamella elegans

Published online by Cambridge University Press:  01 February 2000

Rong-Fu WANG ,
Ashraf A. KHAN ,
Wei-Wen CAO  and
Carl E. CERNIGLIA
Rong-Fu WANG
Affiliation:
Microbiology Division, National Center for Toxicological Research, FDA, Jefferson, AR 72079, USA
Ashraf A. KHAN
Affiliation:
Microbiology Division, National Center for Toxicological Research, FDA, Jefferson, AR 72079, USA
Wei-Wen CAO
Affiliation:
Microbiology Division, National Center for Toxicological Research, FDA, Jefferson, AR 72079, USA
Carl E. CERNIGLIA
Affiliation:
Microbiology Division, National Center for Toxicological Research, FDA, Jefferson, AR 72079, USA
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Abstract

A polyclonal antibody was raised against microsomes prepared from Cunninghamella elegans and used to screen a C. elegans cDNA library. A cDNA clone from this screen was obtained, which corresponds to the C. elegans enolase gene. A single open reading frame (ORF) of 1311 bases was found, which encodes a protein of mol. wt 46882.9. The gene was found to start with an unusual initiation codon TCG, not a standard ATG codon. This was demonstrated first by ORF analysis of the determined sequence and was further confirmed by alignment of the protein with other published enolase sequences. The enolase gene was sub-cloned and over-expressed in Escherichia coli using the expression vector pQE30. Purified recombinant protein from this sub-clone showed the predicted molecular size in SDS–PAGE gel separations. Enolase enzyme activity was also detected that indicating the TCG initiation codon was correct. The C. elegans enolase was found to cluster with other fungal enolases in a phylogenetic analysis. Bacterial enolases and those of animals were separated in different clusters.

Type
Research Article
Information
Mycological Research , Volume 104 , Issue 2 , February 2000 , pp. 175 - 179
Copyright
© The British Mycological Society 2000

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