Cloning of the gene and amino acid sequence for glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides

Amino acid sequencing of glucose 6-phosphate dehydrogenase (Glc6PD) from Leuconostoc mesenteroides yielded sequence for over 75% of the protein. Two oligonucleotides based on the amino acid sequence were used to isolate a partial Glc6PD gene clone (pLmz delta N65), from a pUC9 library, containing 85...

Full description

Saved in:
Bibliographic Details
Published in:The Journal of biological chemistry 1991-07, Vol.266 (20), p.13028-13034
Main Authors: Lee, W T, Flynn, T G, Lyons, C, Levy, H R
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Base Sequence
Cloning, Molecular
Codon - genetics
genes
Genes, Bacterial
glucose-6-phosphate dehydrogenase
Glucosephosphate Dehydrogenase - genetics
Glucosephosphate Dehydrogenase - isolation & purification
Humans
Leuconostoc - enzymology
Leuconostoc - genetics
Molecular Sequence Data
Oligonucleotide Probes
Peptide Fragments - isolation & purification
Polymerase Chain Reaction
Protein Conformation
Recombinant Proteins - isolation & purification
Restriction Mapping
Sequence Homology, Nucleic Acid
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Amino acid sequencing of glucose 6-phosphate dehydrogenase (Glc6PD) from Leuconostoc mesenteroides yielded sequence for over 75% of the protein. Two oligonucleotides based on the amino acid sequence were used to isolate a partial Glc6PD gene clone (pLmz delta N65), from a pUC9 library, containing 85% of the coding sequence and the 3'-untranslated DNA, but lacking the 5'-noncoding DNA sequence and the portion of the gene encoding the 65 N-terminal amino acids. Attempts to obtain a full-length clone from lambda libraries were unsuccessful, possibly due to restriction of L. mesenteroides DNA by Escherichia coli host cells. The 5'-untranslated DNA was amplified by the polymerase chain reaction and partially sequenced. To obtain unmodified DNA for the gene, oligonucleotides corresponding to the 5'- and 3'-noncoding sequences were used to amplify the gene by the polymerase chain reaction, and a 1.8-kilobase pair fragment was isolated and cloned into pUC19. The recombinant plasmid, pLmz, contains the entire Glc6PD gene and expresses the gene in E. coli. pLmz was sequenced showing that the enzyme consists of 485 amino acids. L. mesenteroides Glc6PD is 31% identical to the human enzyme.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(18)98798-3