Cloning and in vitro expression of ferredoxin-I from Capsicum annuum L

Ton, L 2010, Cloning and in vitro expression of ferredoxin-I from Capsicum annuum L, Masters by Research, Applied Sciences, RMIT University.


Document type: Thesis
Collection: Theses

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Title Cloning and in vitro expression of ferredoxin-I from Capsicum annuum L
Author(s) Ton, L
Year 2010
Abstract Ferredoxin is a ubiquitous protein among living organisms involved in many fundamental metabolic processes. The contribution of plant ferredoxin-I to bacterial disease resistance of plants has been proven, with the emphasis on the central role of the iron binding domain. Iron accumulation by ferredoxin-I may play an important role in limiting the growth of invading bacteria. The N-terminal signal peptide of the protein may also have an effect on the anti-microbial activity of the protein by directing the protein into chloroplasts instead of maintaining it in the cytoplasm.

The objective of this study was to clone the ferredoxin gene from Capsicum leaves and investigate the expression of this cloned genes in E. coli. The gene isolation was based on the published nucleotide sequence for the plant ferredoxin-like protein AF03662. Isolated sequences of ferredoxins both with and without signal sequence (Pflp and T-Pflp, respectively) were sub-cloned into pGEM-T Easy vector and DNA sequence was determined. The expression vectors for these gene sequences were constructed with the use of pRSET B expression system. The protein expressions that utilized BL21(DE3)pLysS as the host cells were inducted with 1 mM IPTG for 6 hour before the cell lyse with the sonicator. His-tagged proteins were purified from 6-hour cell lysate through PolyPrep conlumn containing Ni-NTA under native conditions with the use of immidazole. The protein expressions and purifications were evaluated by SDS-PAGE analysis followed by either Coomassie staining and immunodetection.
Degree Masters by Research
Institution RMIT University
School, Department or Centre Applied Sciences
Keyword(s) ferredoxin-I
Capsicum
bacterial disease resistance
Pflip
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Created: Fri, 10 Dec 2010, 15:26:29 EST
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