Expression of human ferredoxin and assembly of the [2Fe-2S] center in Escherichia coli.
A cDNA fragment encoding human ferredoxin, a mitochondrial [2Fe-2S] protein, was introduced into Escherichia coli by using an expression vector based on the approach of Nagai and Thøgersen [Nagai, K. & Thøgersen, M. C. (1984) Nature (London) 309, 810-812]. Expression was under control of the lambda PL promoter and resulted in production of ferredoxin as a cleavable fusion protein with an amino-terminal fragment derived from bacteriophage lambda cII protein. The fusion protein was isolated from the soluble fraction of induced cells and was specifically cleaved to yield mature recombinant ferredoxin. The recombinant protein was shown to be identical in size to ferredoxin isolated from human placenta (13,546 Da) by NaDodSO4/PAGE and partial amino acid sequencing. E. coli cells expressing human ferredoxin were brown in color, and absorbance and electron paramagnetic resonance spectra of the purified recombinant protein established that the [2Fe-2S] center was assembled and incorporated into ferredoxin in vivo. Recombinant ferredoxin was active in steroid hydroxylations when reconstituted with cytochromes P-450scc and P-450(11) beta and exhibited rates comparable to those observed for ferredoxin isolated from human placenta. This expression system should be useful in production of native and structurally altered forms of human ferredoxin for studies of ferredoxin structure and function.
Coghlan
V M
VM
Department of Biological Chemistry, University of California, Irvine 92717.
Vickery
L E
LE
D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D005288:Ferredoxins / Q000235:genetics* / Q000378:metabolism; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D007501:Iron / Q000378:metabolism; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011994:Recombinant Proteins / Q000378:metabolism; D013455:Sulfur / Q000378:metabolism
D004274:DNA, Recombinant; D005288:Ferredoxins; D011994:Recombinant Proteins; D013455:Sulfur; D007501:Iron