Hyperproduction of recombinant ferredoxins in escherichia coli by coexpression of the ORF1-ORF2-iscS-iscU-iscA-hscB-hs cA-fdx-ORF3 gene cluster.
Fe-S proteins acquire Fe-S clusters by an unknown post-translational mechanism. To study the in vivo synthesis of the Fe-S clusters, we constructed an experimental system to monitor the expressed ferredoxin (Fd) as a reporter of protein-bound Fe-S clusters assembled in Escherichia coli. Overexpression of five Fds in a T7 polymerase-based system led to the formation of soluble apoFds and mature holoFds, indicating that assembly of the Fe-S cluster into apoFd polypeptides is a rate-limiting step. We examined the coexpression of the E. coli ORF1-ORF2-iscS-iscU-iscA-hscB-hsc A-fdx-ORF3 gene cluster, which has recently been suggested to be involved in the formation or repair of Fe-S protein [Zheng, L., Cash, V.L., Flint, D.H., and Dean, D.R. (1998) J. Biol. Chem. 273, 13264-13272], with reporter Fds using compatible plasmids. The production of all five reporter holoFds examined was dramatically increased by the coexpression of the gene cluster, and apparent specificity to the polypeptides or to the type of Fe-S clusters was not observed. The increase in holoFd production was observed under the coexpression conditions in all culture media examined, with either 2 x YT medium or Terrific broth, and with or without supplemental cysteine or iron. These results indicate that the proteins encoded by the gene cluster are involved in the assembly of the Fe-S clusters in a wide variety of Fe-S proteins.
Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043, Japan.
M
M
Nakamura
K
K
Saeki
Y
Y
Takahashi
D013437:Carbon-Sulfur Lyases / Q000235:genetics / Q000378:metabolism; D002455:Cell Division / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005288:Ferredoxins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D018628:Gene Dosage; D017930:Genes, Reporter; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D005810:Multigene Family; D016366:Open Reading Frames; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism
D003470:Culture Media; D029968:Escherichia coli Proteins; D005288:Ferredoxins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C095453:HscB protein, E coli; D018832:Molecular Chaperones; D011994:Recombinant Proteins; C085650:hscA protein, E coli; D013437:Carbon-Sulfur Lyases; C080546:cysteine desulfurase