Garba et al
Greener Journal of Biochemistry and Biotechnology Vol. 4 (3), pp. 027-032, September 2017.
2384-6321© 2017 Greener Journals
Manuscript Number: 060117070
Purification and Characterization of 6xHis Tagged Green Fluorescence Protein (GFP)
Nasir Anka Garba1*, Shehu Abdullahi, Kabiru Usman1,
Dayo Oke Okusaga2
1Department of Chemistry, Federal University Gusau, Zamfara State, Nigeria
2Department of Biological Sciences, the University of Essex, Wivenhoe Park, Colchester, UK
The major purpose of this work was to determine if a protein i.e. Green Fluorescent Protein (GFP) could be tagged using an epitope (6xHis tag) through recombinant DNA technology with an expression vector (pET28a) then expressed in E. coli followed by isolation of the protein which was purified using purification steps that not only achieved the high level of purity as desired but was also less time consuming. This was proved by isolation of the recombinant plasmid DNA thus purified with the use of purification columns sequentially i.e. affinity chromatography, gel filtration and ion exchange chromatography, each purification step contributed to the purity attained in the protein which was then tested through SDS-PAGE electrophoresis. The molecular weight of GFP was determined by use of single bands formed by the elution collected from each purification fraction on stained gels through use of coomassie blue stain, the molecular weight was approximated using the protein marker provided (Invitrogen Benchmarker Protein Ladder) and was found to be 27KDa. The protein was further analysed using mass spectrometry for a more accurate molecular mass and for derivation of its structure. The method was found to be effective though a few problems were encountered but the aim of the experiment was successfully achieved.
Keywords: Protein purification, Green Fluorescence Protein, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, recombinant DNA, 6xHis tag, affinity chromatography, gel filtration and ion exchange chromatography.
Cubbit, A., Heim, R., Adams, S., Boyd, A., Gross, L., and Tsien, R. (1995). Understanding, improving and using green fluorescent proteins. TIBS 20:448-55.
Morin, J., and Hastings, J. (1971). Energy transfer in a bioluminescent system. J. Cell Physiol, 77:313-8.
Sambrook, J., and Russell, D. W. (2001.) Molecular Cloning: A Laboratory Manual. 3rd Edition, Cold Spring Harbor Laboratory Press, New York, USA, 5:5.1-5.48
Kahana, J., and Silver, P. (1996) in Current Protocols in Molecular Biology, F.Ausabel,et al, Editors. Green and Wiley: New York. p 9.7.22-9.7.28.
Moores, S., Sabry, J., and Spudich. J. (1996). Myosin dynamics in live Dictyostelium cells. Proc Nati Acad Sci, USA. 93: 443-446.
Olsen, K., McIntosh, J. and Olmstead, J. (1995). Analysis of MAP4 function in living cells using green fluorescent protein (GFP) chimeras. J Cell Biol 130: 639-650.