Purify GFP From Aequorea Victoria

Purify GFP From Aequorea VictoriaMethods and Results GFP was cloned into E. coli strain JM109 and expressed under optimum conditions in Luria broth agar containing ampicillin and IPTG for induction. Protein was extracted by lysis using form milling proficiency and fluorescence of protein measured in a fluorimeter, concentration of both pure and crude proteins were obtained with Bradford (1976) method. Purity of GFP was further confirmed by SDS-PAGE stain with coomassie blue.Conclusion ad hoc activity(RFUmg-1) of pure protein increased compared to crude representing increase in purity, with a substantial yield of 82%.Significance and Impact of news report This study proved Ion exchange chromatography as a reliable technique for GFP purification and blue percentage recovery for use as a reporter gene in molecular(a) biology studies.Keywords GFP, purity, Ion exchange chromatography, Specific activity, fluorescence.INTRODUCTIONThe jelly lean Aequorea capital of Seychelles, emits a bluish light from the delimitation of its umbrella (Inuoye, and Tsuji 1994). The light is produced by the bioluminescent jellyfish when calcium binds to the photoprotein aequorin. Although activation of aequorin in vitro or in heterologous cells produces blue light, the jelly fish produces green light. This light is the result of a second protein in A. victoria that derives its excitation energy from aequorin, the green fluorescent protein (Chalfie et al., 1994).Green fluorescent protein (GFP) is a protein of 238 amino acid residues. It is a highly perpetual protein possessing a tightly packed can tertiary structure that is resistant to many biological denaturants, most proteases, pH (5-12), temperature (Tm=78 C), and chaotropic salts (McRae et al., 2005 Zhuang, et al., 2008). Purified GFP absorbs blue light (maximally at 395nm with a minor peak at 470nm) and emits green light (peak emission at 509nm with a shoulder of 540nm). This fluorescence is stable and virtually no photobl eaching is observed (Chalfie et al., 1994). The stable and intense fluorescence of GFP without any cofactors in many different organisms makes is ideal for molecular biology applications such as markers for gene expression analysis of molecular interactions and also as biological information storage devices and optical biosensors in areas of Nanotechnology (McRae et al., 2005).Current purification procedures, specific for GFP include multiple phase high performance liquid chromatography (HPLC), chromatofocusing on a pH gradient, metal ion precipitancy and organic extraction. Most of these methods are either expensive, time consuming or give low yields with