Expression of human ferredoxin and assembly of the [ 2 Fe2 S ] center in Escherichia coli ( mitochondrial iron-sulfur protein / electron transfer / adrenodoxin )

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Author	Coghlan, Vincent M. Vickery, Larry E.
Abstract	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 Th0gersen [Nagai, K. & Thogersen, M. C. (1984) Nature (London) 309, 810-812]. Expression was under control of the A PL promoter and resulted in production of ferredoxin as a cleavable fusion protein with an aminoterminal fragment derived from bacteriophage AcIM 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-450c and P-45011p 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. The vertebrate ferredoxins are small (13-14 kDa) iron-sulfur proteins that occur in the mitochondria of steroid metabolizing tissues (1-4). They function to transfer reducing equivalents from NADPH-oxidoreductases to cytochrome P-450 enzymes involved in the biogenesis of steroid hormones, the production of bile acids, and the formation of vitamin D metabolites. Ferredoxins play a central role at regulated steps in these processes, and it is of particular interest to identify structural features that determine the properties of the iron-sulfur center and the specific interactions with steroid hydroxylase components. The ferredoxin from bovine adrenal cortex, designated adrenodoxin, has been most extensively characterized, primarily because of its relative abundance in this tissue (5-8). Bovine adrenodoxin is synthesized in the cytoplasm with a large amino-terminal presequence, which is proteolytically removed upon mitochondrial uptake (9-12). A single [2Fe2S] center is incorporated into the apoprotein, presumably within mitochondria, to produce the active holoprotein; it is not known whether incorporation of the prosthetic group occurs before or after removal of the presequence. Recently, we isolated ferredoxin from human placental mitochondria and showed that it is structurally and functionally similar to bovine adrenodoxin (13). Sequence analysis of human ferredoxin and of specific cDNAs from a human placental library revealed that the protein is synthesized as a 19,371-Da precursor that is processed to mature protein of 13,546 Da (13, 14). Complementary DNA from a human adrenal library (15) is identical to that found in the placenta, suggesting that a single gene product may function in all steroidogenic tissues within an organism. Ferredoxin structure must therefore be compatible with a variety of steroid hydroxylase components. Unfortunately, it is difficult to obtain human ferredoxin in amounts required for most structural analyses because of the low level present in placenta (13) and the lack of availability of sufficient amounts of other steroidogenic tissues. In this report, we describe a system for high-level expression of human ferredoxin cDNA in E. coli. Using the approach developed by Nagai and Thogersen (16), human ferredoxin is produced as a stable fusion protein with the bacteriophage AcIl protein; the proteins are linked via a peptide, which includes a region that is recognized and cleaved by factor Xa, a sequence-specific protease. The ferredoxin fusion protein produced is soluble, and a [2Fe2S] center is incorporated in vivo. Specific cleavage of the fusion protein yields fully active ferredoxin that is indistinguishable from the placental protein. This system allows for future investigations into the assembly and incorporation of the ferredoxin iron-sulfur center and provides a means by which mutant forms of the protein can be obtained for studies of ferredoxin structure and function. MATERIALS AND METHODS Materials. Restriction endonucleases and other enzymes used in plasmid construction were obtained from Boehringer Mannheim unless noted otherwise; reaction conditions were as specified by the supplier. Reagents for bacterial growth media were from Difco, and other chemicals were from Sigma. Construction of pHFdxl. A 397-base-pair (bp) fragment containing a portion of human ferredoxin coding sequence was obtained by Apa I and Xmn I digestion of plasmid from a previously isolated cDNA clone (14). The fragment was treated with 10 units of mung bean nuclease (Stratagene) per pgg of cDNA at 370C for 30 min to produce blunt-ended fragments of various lengths. The plasmid pfX8 was provided by Charles Glabe (University of California, Irvine) and contains the factor Xa linker from M13mp11FX (16) inserted into the BamHI site of pUC8. Plasmid pfX8 was linearized with Stu I and Pst I and treated with mung bean nuclease as described above, except 1 unit per ,Ag of DNA was used. Blunt-ended pfX8 was dephosphorylated with alkaline phosphatase by using 12 units per ug ofDNA for 30 min at 370C. The dephosphorylated pfX8 was mixed and ligated to a 3-fold excess of blunt-ended ferredoxin cDNA fragments. The ligation mixture was used to transform competent E. coli HB101, and recombinants were screened on lifts with 32pAbbreviation: EPR, electron paramagnetic resonance. 835 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 836 Biochemistry: Coghlan and Vickery labeled nick-translated ferredoxin cDNA (14). Plasmids were isolated (17) from selected positive colonies and mapped with various restriction endonucleases to select those having proper orientation of the ferredoxin sequence. BamHI and HindIII digestion of these plasmids produced fragments containing the factor Xa site-human ferredoxin fusion sequence. These fragments were isolated, ligated into BamHI/HindlIl-cleaved M13mpl9, and sequenced by the dideoxy method of Sanger et al. (18). A fragment was identified that had the codon for the first serine of mature ferredoxin immediately adjacent to the 3' end of the factor Xa recognition sequence. This fragment was ligated into pMb3 (19) that had been cleaved with BamHI and HindIll, and the mixture was used to transform E. coli HB101 cells. Plasmid isolated from transformed cells was designated pHFdxl. Expression and Purification of Protein. Competent E. coli MZ-1 cells, a defective lysogenic strain (16), were transformed (17) with pHFdxl. Transformed cells were grown overnight at 30'C in 1 ml of Luria-Bertani medium (17) supplemented with ampicillin (50 Ag/ml) (LB-amp). This culture was used to inoculate 1 liter of LB-amp in a 2-liter flask, and cells were grown at 30'C to an OD6w of -0.7. The culture was then heated in a 650C water bath with rapid swirling to increase the temperature to 420C and to induce synthesis of the ferredoxin fusion protein. The culture was incubated at 420C for 2 hr and at 370C for an additional 18 hr. All subsequent steps were performed at 4°C. Cells were harvested by centrifugation at 4000 x g for 5 min. The cellular pellet was resuspended in 40 ml of 50 mM Tris HCl (pH 7.6) containing 0.1 mM EDTA, and the cells were lysed by two passages through a French press (10,000 psi; 1 psi = 6.9 kPa). Cellular debris was removed from the lysate by centrifugation at 35,000 x g for 15 min, yielding a straw-brown supernatant fluid. The ferredoxin fusion protein was purified from the soluble extract as described for human placental ferredoxin (13). Briefly, the supernatant was loaded onto a DEAE-cellulose column and washed, and the brown ferredoxin-containing band was removed to a separate column and eluted with sodium chloride. The preparation was subjected to further chromatography on DEAE-agarose and Sephadex G-75. The purified fusion protein was treated with human factor Xa protease (Boehringer Mannheim) at a ratio of 1:50 (wt/wt) for 4 hr at 25°C in 50 mM Tris-HCl (pH 7.8) containing 0.1 mM EDTA. The processed mature ferredoxin was purified by ion-exchange chromatography on DEAE-agarose (2.5 x 20 cm) followed by gel filtration on Sephadex G-75 (1.0 x 40 cm). Analytical Methods. Samples were analyzed by NaDodSO4/PAGE using 15% gels and the buffer system of Laemmli (20). Protein concentrations were determined by the method of Lowry et al. (21). Gels were electroblotted onto polyvinyl difluoride membranes (Millipore) and stained with Coomassie blue R-250. N-terminal amino acid sequences were determined by William T. Morgan (Bio-Technologies Unit, Louisiana State University Medical School, New Orleans) using automated Edman degradation on an Applied Biosystems 477A sequenator. Samples to be sequenced were denatured by boiling in NaDodSO4/PAGE sample buffer, vacuum-blotted onto polyvinyl difluoride membranes, stained with Coomassie dye, and excised for direct analysis. C-terminal sequencing was performed with carboxypeptidase P as described (13). Electron paramagnetic resonance (EPR) spectra were recorded by Gerard Jensen and Phillip Stevens (University of Southern California, Los Angeles) using a Bruker ESR-200D spectrometer equipped with an Oxford Instruments ESR-9 flow cryostat. An anaerobic 100 ,M sample in 50 mM Tris-HCI, pH 7.4/0.1 mM EDTA was treated with 3 mM sodium dithionite. Spectra were obtained from 10-60 K at 9.5956 GHz using 6.3 kkW power and S G modulation amplitude at 100 kHZ. Absorption spectra were recorded at ambient temperature in a Cary 17D spectrophotometer
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