Overproduction of a selenocysteine-containing polypeptide in Escherichia coli: the fdhF gene product.
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 selenopolypeptide 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 75Se, 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.
Chen
G T
GT
Department of Biochemistry, Robert Wood Johnson Medical School, Rutgers, University of Medicine and Dentistry of New Jersey, Piscataway 08854.
Axley
M J
MJ
Hacia
J
J
Inouye
M
M
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D006864:Hydrogenase / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics*; D016566:Organoselenium Compounds / Q000378:metabolism*; D010957:Plasmids; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics; D012643:Selenium / Q000378:metabolism; D017279:Selenocysteine
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D009097:Multienzyme Complexes; D016566:Organoselenium Compounds; D012345:RNA, Transfer, Amino Acid-Specific; C036928:tRNA, selenocysteine-; D017279:Selenocysteine; D006864:Hydrogenase; D005560:Formate Dehydrogenases; C054017:formate hydrogenlyase; D012643:Selenium; D003545:Cysteine