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Expression of P450fas in E. coli: Role in the Biosynthesis of Plant Cytokinin Zeatin
Author(s) -
Pastore Mayra,
Gerber Nancy C.,
Wang Yu
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a459-c
Subject(s) - zeatin , operon , biology , cytokinin , ferredoxin , plasmid , gene , virulence , biosynthesis , escherichia coli , microbiology and biotechnology , biochemistry , enzyme , auxin
Rhodoccocus Fascians is phytopathogenic bacteria able to induce leafy galls through the expression of the virulent promoting Fas Operon . The operon, located within the Fi plasmid, codes, among other proteins, Cytochrome P450 fas , and its ferredoxin‐like partner, Fasredoxin (FasRdx). Both proteins are essential for plant fasciation. The proposed mechanism for infection places P450 fas in the biosynthesis pathway of the cytokinin‐zeatin, a plant hormone. In order to study this proposal, we have cloned the genes coding for polyhistidine‐tagged P450 fas and FasRdx into the pCWori vector and expressed the proteins in E. coli. We have obtained positive results for P450 fas expression after running samples through SDS‐PAGE, Western blotting, UV‐Visible spectrometer, and DNA sequencing. Thus far, we have successfully purified P450 fas and shown it is able to bind to cytokinin precursors such as isopentenyladenine. However, our efforts have been shortcoming for expressing FasRdx and we are currently screening for better cell growth conditions and the used of engineered E. coli cell line (Rosetta TM 2(DE3)pLysS TM , Stratagene), shows promise of producing functional protein. Founded by: Minority Access to Research Careers (MARC)