Premium
Overproduction of a selenocysteine‐containing polypeptide in Escherichia coli : the fdhF gene product
Author(s) -
Chen Giafen T.,
Axley Milton J.,
Hacia Joseph,
Inouye Masayori
Publication year - 1992
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1992.tb01528.x
Subject(s) - selenocysteine , biology , formate dehydrogenase , escherichia coli , gene product , gene , biochemistry , plasmid , microbiology and biotechnology , selenoprotein , protein subunit , amino acid , formate , gene expression , cysteine , glutathione , enzyme , glutathione peroxidase , catalysis
Summary The fdhF gene of Escherichia coli codes for the selenocysteine‐including protein subunit of formate dehydrogenase H. The protein subunit consists of 715 amino acid residues containing a single selenocysteine residue at position 140 which is encoded by a UGA codon. The decoding of this opal termination codon occurs under anaerobic growth conditions by means of a specific tRNA, i.e. the selC gene product. The ability of E. coli cells to overproduce a seleno‐polypeptide was examined using the fdhF gene as a model system. Surprisingly, E. coli was able to synthesize the fdhF gene product at the level of approximately 12% of the total cellular protein. This was achieved by cloning fdhF in a multicopy plasmid together with a synthetic selC gene under the Ipp promoter. FdhF production was absolutely dependent upon the addition of selenium to the culture medium and was almost completely blocked in the presence of oxygen. The product was specifically labelled with 75 Se, proving that it consisted of a selenoprotein. The product was purified to homogeneity and shown to exhibit the catalytic properties characteristic of formate dehydrogenase H.