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[Isolation and sequencing of a novel form of cytochrome p-450 4F family from human liver].

OBJECTIVE
To isolate and sequence a novel gene of cytochrome p-450 4F family.


METHODS
A cDNA was isolated by touchdown PCR from a cDNA library of human fetal liver using specific primers. The DNA sequencing was done by an auto-sequence machine through forward and reverse directions. The expression of cDNA encoded protein was tested in yeast cells.


RESULTS
Sequencing of the full length cDNA revealed a sequence of 1,980 nucleotides coding for a protein of 520 amino acids with a molecular weight of 59,700 Da. The cDNA-deduced amino acid sequence showed 92% homology to that of human polymorphonuclear leukocytes (PMN) LTB4-hydroxylase (CYP4F3) and 91% homology to that of human liver LTB4 hydroxylase (CYP4F2). The expression of protein in the yeast cells showed a fusion protein with a molecular weight of about 86,000 Da and 60,000 Da after a proteolytic removal of the GST affinity tag. It was confirmed by RT-PCR that the gene was expressed in the adult liver.


CONCLUSION
Our results clearly show a novel form of cytochrome p-450 4F in human liver.