Greener Journal of Biochemisty and Biotechnology

Greener Journal of Biochemistry and Biotechnology Vol. 4 (3), pp. 027-032, September 2017.

  2384-6321© 2017 

Research Paper

Manuscript Number: 060117070

(DOI: http://doi.org/10.15580/GJBB.2017.3.060117070)

 

Purification and Characterization of 6xHis Tagged Green Fluorescence Protein (GFP)

 

Nasir Anka Garba^1*, 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

Abstract

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.

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