Expression of human ferredoxin and assembly of the (2Fe-2S) center in Escherichia coli
- Univ. of California, Irvine (USA)
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 Thogersen. Expression was under control of the {lambda}P{sub L} 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 NaDodSO{sub 4}/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-450{sub sec} and P-450{sub 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.
- OSTI ID:
- 7068690
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America; (USA), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (USA) Vol. 86:3; ISSN PNASA; ISSN 0027-8424
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ABSORPTION SPECTROSCOPY
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CELL CONSTITUENTS
DAYS LIVING RADIOISOTOPES
DNA
DNA SEQUENCING
DNA-ASE
ELECTRON TRANSFER
ELECTROPHORESIS
ENDONUCLEASES
ENZYMES
ESCHERICHIA COLI
ESTERASES
FERREDOXIN
GENE REGULATION
HYDROLASES
ISOTOPES
LIGHT NUCLEI
METALLOPROTEINS
MICROORGANISMS
MITOCHONDRIA
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANOIDS
PHOSPHODIESTERASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PROTEINS
RADIOISOTOPES
RECOMBINANT DNA
SPECTROSCOPY
STRUCTURAL CHEMICAL ANALYSIS