Purify GFP From Aequorea Victoria

Purify GFP From Aequorea Victoria Methods and Results: GFP was cloned into E. coli strain JM109 and expressed under optimal conditions in Luria broth agar containing ampicillin and IPTG for induction. Protein was extracted by lysis using bead milling technique 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 stained with coomassie blue. Conclusion: Specific activity(RFUmg-1) of pure protein increased compared to crude representing increase in purity, with a substantial yield of 82%. Significance and Impact of Study: This study proved Ion exchange chromatography as a reliable technique for GFP purification and high percentage recovery for use as a reporter gene in molecular biology studies. Keywords: GFP, purity, Ion exchange chromatography, Specific activity, fluorescence. INTRODUCTION The jelly fish Aequorea victoria, emits a bluish light from the margin 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 stable protein possessing a tightly packed ÃŽ ²ÃƒÅ'†°ÃƒÅ'†°ÃƒÅ'†°ÃƒÅ'†°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 photobleaching 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 precipitation and organic extraction. Most of these methods are either expensive, time consuming or give low yields with