Identification and sequencing of a cDNA encoding 6-phosphogluconate dehydrogenase from a fungus,Cunninghamella elegansand expression of the gene inEscherichia coli
| Autor: | Carl E. Cerniglia, Wei-Wen Cao, Rong-Fu Wang, Ashraf A. Khan |
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| Rok vydání: | 1998 |
| Předmět: |
DNA
Complementary Genetic Vectors Molecular Sequence Data Dehydrogenase Sequence alignment Biology Molecular cloning Polymerase Chain Reaction Microbiology law.invention Fungal Proteins Open Reading Frames law Complementary DNA Genetics Amino Acid Sequence Cloning Molecular Phosphogluconate dehydrogenase Molecular Biology Gene Cunninghamella cDNA library Phosphogluconate Dehydrogenase Molecular biology Recombinant Proteins Biochemistry Genes Bacterial Recombinant DNA Electrophoresis Polyacrylamide Gel Sequence Alignment |
| Zdroj: | FEMS Microbiology Letters. 169:397-402 |
| ISSN: | 1574-6968 0378-1097 |
| DOI: | 10.1111/j.1574-6968.1998.tb13346.x |
| Popis: | The fungus, Cunninghamella elegans has been widely used in bioremediation and microbial models of mammalian studies in many laboratories. Using the polymerase chain reaction to randomly amplify the insert directly from the single non-blue plaques of a C. elegans cDNA library, then partly sequencing and comparing with GenBank sequences, we have identified a clone which contains C. elegans 6-phosphogluconate dehydrogenase gene. The polymerase chain reaction product was cloned into a plasmid, pGEM-T Easy vector for full insert DNA sequencing. The 6-phosphogluconate dehydrogenase gene (1458 bases) and the deduced protein sequence were determined from the insert DNA sequence. The gene was found by open reading frame analysis and confirmed by the alignment of the deduced protein sequence with other published 6-phosphogluconate dehydrogenase sequences. Several highly conserved regions were found for the 6-phosphogluconate dehydrogenase sequences. The 6-phosphogluconate dehydrogenase gene was subcloned and over-expressed in a plasmid-E. coli system (pQE30). The cell lysate of this clone has a very high 6-phosphogluconate dehydrogenase enzyme activity. Most of the recombinant protein in this system was formed as insoluble inclusion bodies, but soluble in high concentration of urea-buffer. Ni-NTA resin was used to purify the recombinant protein which showed 6-phosphogluconate dehydrogenase enzyme activity. The recombinant protein has a predicted molecular size correlating with that revealed by sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. The C. elegans 6-phosphogluconate dehydrogenase was in a cluster with yeast' 6-phosphogluconate dehydrogenase in the phylogenetic tree. Bacterial 6-phosphogluconate dehydrogenase and higher organisms' 6-phosphogluconate dehydrogenase were found in different clusters. |
| Databáze: | OpenAIRE |
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