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Over‐expression of Escherichia coli F 1 F o –ATPase subunit a is inhibited by instability of the uncB gene transcript
Author(s) -
Arechaga Ignacio,
Miroux Bruno,
Runswick Mike J.,
Walker John E.
Publication year - 2003
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(03)00677-x
Subject(s) - escherichia coli , microbiology and biotechnology , protein subunit , gene , biology , gene expression , messenger rna , fusion protein , fusion gene , atpase , northern blot , rna polymerase , biochemistry , enzyme , recombinant dna
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.