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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.
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 βÌ‰Ì‰Ì‰Ì‰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 <90% purity (Deschamps et al., 1995; McRae et al.,2005).
This present report is an attempt to extract GFP with a procedure that gives higher yield, and a highly pure protein. The finding that the expressed protein is fluorescent suggests that the primary structure of the protein undergoes modification to form a chromophore during expression. Because of the marked intrinsic fluorescence of the protein, it may also serve as a reporter or marker in gene expression studies (Inuoye and Tsuji, 1994).
MATERIALS AND METHODS
Bacterial strain, plasmid and reagents. Escherichia coli JM109 genotype- e14-(McrA-) recA1 endA1 gyrA96 thi-1 hsdR17 (rK- mK+) supE44 relA1 (lac-proAB) [F´ traD36 proAB lacIqZΔM15] (Stratagene, CA USA), plasmid pHG165 (Stewart et al., 1986) were used for cloning and expressing. Luria bertani was used for optimum growth of E. coli at 37°C overnight, glass beads (Sigma Aldrich Inc, Chemie GMBH Germany), was used to lyse the transformants. Ligation mixture and all chemicals used for this experiment were also supplied as a kind gift from Dr. Phil Hill of the Division of Food Sciences, University of Nottingham, UK.
Ligation mixture. Plasmid pHG165 was constructed from the gene of pBR322 (Stewart et al., 1986).cDNA was prepared from the fluorescent tissue of the jelly fish Aequorea victoria which was used as a template for the PCR amplification of the Gfp transcripts from this mixture. The PCR primers had non-homologous 5' extensions to allow introduction of unique restriction sites at the 5' and 3' ends of the PCR amplicon. Amplicons were cut with EcoRI and PstI and ligated with DNA ligase into the vector pHG165 which were previously cut with the same restriction enzymes. The restriction sites were orientated to achieve directional cloning with the ORF of the Gfp gene downstream of the lacZ promoter (Placz).
Protein expression and extraction. E. coli JM109 was transformed with the ligation mixture (20µl) using the CaCl2 transformation method (Sambrook and Russell, 2001.). LB broth(750 µl) was added to the transformed cells and incubated for 30min before spreading on LB agar containing 0.5mMml-1 IPTG (Isopropylthiogalactoside) for induction, 50µgml-1 ampicillin and incubated overnight under aerobic conditions at 37°C. Transformants were selected from LB plates through the green fluorescent phenotypic traits of the colonies and lysed using the bead milling technique. 10ml of liquid culture was centrifuged (17,900g, 3min), pellets resuspended in 1.5ml of 20mM cold Tris HCl pH 7.4, and centrifuged, 0.5g of glass beads (102Ao) added and whirl mixed for 4min.Tube was cooled on ice for 5min to allow the beads settle, all the supernatant is transferred into a new tube. 700 µl was then transferred to a fresh tube and stored at -20°C as the crude lysate of the protein.
Protein Purification by Ion exchange chromatography. This was carried out using Diethylaminoethyl (DEAE) sepharose (Sigma Aldrich, Germany) 50mM, 100mM, 150mM, 200mM, and 250mM NaCl, 20mM Tris-HCl, pH 7.4 according to standard procedure (Sambrook and Russell, 2001). All fractions were collected and kept on ice and only the fractions which most fluoresces green were stored at -20 °C.
SDS-PAGE. Crude lysate and fractions were electrophoresed in SDS-PAGE with 12.5% separating gel, and 4% stacking gel according to Laemmli, 1970.
GFP Quantification. Protein concentrations were measured with the Bradford's method (1976) .Fluorescence values were obtained with 50µl of the crude and pure GFP samples each with a fluorescence reader (Tecan Genios Pro Instrument, Germany) at 485nm(emission) and 535nm(excitation).
Expression and Yield of GFP. Plasmid pHG165 expressed GFP in strain JM109 with a transformation efficiency of......... µg-1 DNA. The efficiency of the transformation process using the CaCl2 method gave considerable amount of transformants indicating that gfp was inserted in the plasmid and expressed. Concentration of crude protein after lysis was found to be 0.75mgml-1 , purification was executed in an ion exchange with a concentration of 0.48mgml-1 for the purified fraction. Purification gave a yield of approximately 82% with a loss of 34% in the concentration of the crude protein after purification. Specific activity of crude and purified proteins was determined in a fluorescence reader to be 7.85 x 105 RFUmg-1 and 1.1 x 106 RFUmg-1 respectively. Specific activity of pure protein increased with purification with the highest specific activity at the highest concentration of buffer (250mM NaCl, 20M Tris-HCl) used to elute the crude lysate, suggesting a more purified protein. The RFU ml-1 determined from the fluorescence spectrophotometer reader gave values of 5.9 x 105 and 4.8 x 105 for crude and pure GFP respectively(Table1).The RFUml-1 of the crude decreases as its purified with an increase in the specific activity of purified fractions as suggested above.
Purification. Gfp was purified to homogeneity as confirmed by SDS-PAGE electrophoresis. The single bands (Fig1) for the samples( lanes 5-7) with corresponding relative molecular weight of 28KDa as determined from the standard curve (Fig2) corresponds to the relative molecular weight of pure Gfp protein. Lanes 5-7 represent different eluted fractions fom the crude lysate (150, 200, 250mM NaCl, 20M Tris-HCl). The resolution of the band in lane 7 was better which demonstrated the presence of a more purified protein consistent with the predicted molecular weight of Gfp (28KDa). Taken together, these data indicated that production of Gfp in E. coli JM109 gave considerable amounts of the pure protein that displayed identical properties of native Gfp in A. victoria.
Green fluorescent protein (GFP) is commonly used as a reporter protein in a wide range of biological experiments (Tsien, 2000). Purification of GFP has been carried out in both prokaryotic and eukaryotic expression systems including plants. Cloning of GFP into plasmid vectors has been successful owing to the ability of the protein to withstand extreme conditions such as high temperature and pressure. Current purification techniques for GFP are expensive, and result in high losses of GFP or may be cheap and yet result in low protein yields such as organic extraction methods. In this work, purification of GFP from strain JM109 in an Ion exchange Column Chromatography is adapted. This assay is simple, affordable, high purity, and yield, applying standard laboratory reagents. Though GFP was expressed with E.coli, other workers' have expressed it also using eukaryotes (Papakonstantiou et al., 2009). This study has also shown the efficiency of purification using ion exchange chromatography as a good technique in purification of this important reporter gene.
Carrying our purification techniques such as immobilized metal affinity chromatography involves the fusion of a peptide tag and gives highly pure protein with polyhistidine tags which are difficult to separate, from improperly folded proteins tags, lower yields and its very expensive. Similar problems are faced when using size exclusion chromatography another tag based protocol though they provide higher purity compared to Ion exchange (Papakonstantiou et al., 2009). However with this technique described here, the challenges encountered in previous techniques have been overcome and a pure protein with purity of 1.1 x 106 RFUmg-1 at a concentration of 0.48mgml-1 compared to the crude protein with 7.85 x 105 RFUmg-1 (0.75mg/ml), with a yield of approximately 82%. Further analysis to confirm the purity of GFP using SDS-PAGE analysis with commasie blue staining technique showed bands consistent to the molecular weight of GFP (28KDa) as single bands in the gel suggesting homogeneity of the protein. Purification of Gfp with a two step protocol using affinity chromatography combined with monoclonal antibodies has been demonstrated to provide a better purity and though yield is lower (Zhuang, et al., 2008). Hydrophobic interactions in a column has also been demonstrated in Gfp purification by cmbining it with an initial purification step involving low heat and ammonium sulphate which also demonstrates over 85% purity though the yield is reduced (McRae et al.,2005).
Ion exchange chromatography has been successfully used in this study to purify GFP expressed by JM109 strain of E. coli with a yield of 82%. This study has been able to demonstrate that the specific activity of the protein increases with further purification demonstrating high purity, which can be improved upon by coupling with another purification protocol. Since Gfp is highly tolerant to extreme conditions, new and improved purification protocols can be designed to harness some of these properties.
This work was funded by the School of Biosciences, University of Nottingham. The authors would like to thank Dr. Phil Hill, University of Nottingham for the donation of the Ligation mixture, and the Division of Food Sciences for the supply of all reagents and equipment used.