Amplification of the respiratory NADH dehydrogenase of Escherichia coli by gene cloning.
A relatively simple method has been used to clone the gene coding for the respiratory NADH dehydrogenase (NADH-ubiquinone oxidoreductase) of Escherichia coli from unfractionated chromosomal DNA. The restriction endonucleases EcoRI, BamI and HindIII were used to construct three hybrid plasmid pools from total E. coli DNA and the amplifiable plasmids pSF2124 and pGM706. Three different restriction endonucleases were used to increase the chances of cloning the ndh gene intact. Mobilization by the plasmid F was used to transfer the hybrid plasmids into ndh mutants and selection was made for Apr and complementation of ndh. DNA fragments complementing ndh were isolated from both the EcoRI and HindIII hybrid plasmid pools. The strain carrying the hybrid plasmid constructed with EcoRI produced about 8--10 times the normal level of the respiratory NADH dehydrogenase in the cytoplasmic membrane. Treating the cells with chloramphenicol to increase the plasmid copy number allowed the level of NADH dehydrogenase in the membrane to be increased to 50--60 times the level in the wild type. The results indicate the potential of gene cloning for the specific amplification of particular proteins prior to their purification.
D004261:DNA Replication; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D005796:Genes; D008722:Methods; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics*; D010957:Plasmids
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D009247:NADH, NADPH Oxidoreductases; D004262:DNA Restriction Enzymes