|Title||Over-expression of Escherichia coli F1F(o)-ATPase subunit a is inhibited by instability of the uncB gene transcript.|
|Publication Type||Journal Article|
|Year of Publication||2003|
|Authors||Arechaga, I, Miroux, B, Runswick, MJ, Walker, JE|
|Date Published||2003 Jul 17|
|Keywords||Bacterial Proton-Translocating ATPases, Blotting, Northern, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Gene Expression Regulation, Enzymologic, Kinetics, Protein Denaturation, Protein Folding, Protein Subunits, Proton-Translocating ATPases, Transcription, Genetic|
Little is known about the stability of transcripts encoding membrane proteins in strong expression systems and its effect on membrane protein over-production. We have expressed all the genes encoding subunits of the membrane domain F(o) of the ATP synthase in a T7 RNA polymerase-based system. All of them but uncB (subunit a) were expressed separately at very high levels in the bacterial hosts Escherichia coli C41(DE3) and C43(DE3). However, expression of uncB was extremely toxic to the bacteria. Northern blot analysis showed that the level of accumulation of the mRNA from uncB was very low. Deletion of uncB in combination with gene fusion experiments demonstrated that the middle region of the gene, encoding amino acids 92-171, exhibited a dominant toxic phenotype associated with a very poor level of expression. Green fluorescent protein fusions with N- and C-ends of uncB helped to stabilize the mRNA and to obtain high yields of protein.
|Alternate Journal||FEBS Lett.|