| Budget Amount *help |
¥13,100,000 (Direct Cost: ¥13,100,000)
Fiscal Year 1998: ¥4,200,000 (Direct Cost: ¥4,200,000)
Fiscal Year 1997: ¥8,900,000 (Direct Cost: ¥8,900,000)
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| Research Abstract |
Halaarcula japonica strain TR-1 is a predominantly triangular disk-shaped, extremely halophilic archdeacon. We have purified a novel ferredoxin (Fd) from Ha. japonica to electrophoretic homogeneity by anion exchange chromatography and gel filtration. It had an apparent molecular mass of 24 kDa on SDS-polyacrylamide gel electrophoresis. Amino acid composition analysis revealed that the Ha. japonica Fd contained many acidic amino acids. The N-terminal amino acid sequence was determined to be H2N-PTV-EYLNYEVVDDNGWDMYDDDVFAEASDM.The absorption and ESR spectra of the Ha. japonica Fd revealed that the Fd contained a [2Fe-2S] cluster. Polymerase chain reaction (PCR) was performed using the genomic DNA of Ha. japonica as a template. The sense primer was designed from the N-terminal amino acid sequence of the Ha. japonica Fd ; the antisense primer was based on the conserved polypeptide region among other halobacterial Fds. The DNA fragment of about 160 bp, presumed to be a part of the Fd gene,
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was amplified by PCR and used as a probe in Southern hybridization analyses of genomic DNA.Genomic DNA digested with Sal I showed a hybridization band at 3.1 kb. A subgenomic library, I containing Sal I digests of about 3 kb, was constructed and screened for the Fd gene by colony hybridization. A positive clone was obtained and Found to contain a recombinant plasmid with a 3.1-kb Sal I insert. The nucleotide sequence of the 3.1-kb fragment revealed an open reading frame of 387 nt coding for a polypeptide of 129 amino acids. The deduced amino acid sequence at the N-terminal region is in agreement with that determined by peptide sequencing, assuming a processing the initial methionine. The amino acid sequence showed about 43%, 98% and 87% identities with those of spinach, Haloarcula marismortui and Halobacterium salinarium Fds, respectively. Fds from Ha. Japonica and other halobacteria had an additional 20 a.a. in the N-terminal region compared to the spinach Fd. This N-terminal region seemed to play an important role for halotolerance. Next, the Ha. japonica Fd gene was ligated to the expression plasmids pET-2[d(*) and pGEX-5X-1, and then transformed to Escharichia coli. The recombinant Fds were successfully produced as fusion proteins with T7tag or glutathione 5-transferase. Both Fd fusions, however, did not contain Fe-S clusters. Less
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