Open AccessCloning, expression, and characterization of the Anabaena thioredoxin gene in Escherichia coli
Author(s) -
ChangJin Lim,
Florence K. Gleason,
James A. Fuchs
Publication year - 1986
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.168.3.1258-1264.1986
Subject(s) - biology , thioredoxin , escherichia coli , gene , microbiology and biotechnology , peptide sequence , anabaena , molecular cloning , ferredoxin thioredoxin reductase , heterologous expression , biochemistry , cloning (programming) , recombinant dna , genetics , thioredoxin reductase , bacteria , cyanobacteria , computer science , programming language
The gene encoding thioredoxin in Anabaena sp. strain PCC 7119 was cloned in Escherichia coli based on the strategy that similarity between the two thioredoxins would be reflected both in the gene sequence and in functional cross-reactivity. DNA restriction fragments containing the Anabaena thioredoxin gene were identified by heterologous hybridization to the E. coli thioredoxin gene following Southern transfer, ligated with pUC13, and used to transform an E. coli strain lacking functional thioredoxin. Transformants that complemented the trxA mutation in E. coli were identified by increased colony size and confirmed by enzyme assay. Expression of the cloned Anabaena thioredoxin gene in E. coli was substantiated by subsequent purification and characterization of the algal protein from E. coli. The amino acid sequence derived from the DNA sequence of the Anabaena gene was identical to the known amino acid sequence of Anabaena thioredoxin. The E. coli strains which expressed Anabaena thioredoxin complemented the TrxA- phenotype in every respect except that they did not support bacteriophage T7 growth and had somewhat decreased ability to support bacteriophages M13 and f1.