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Engineering Green Fluorescent Protein as a Dual Functional TAG

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Item Summary

Title: Engineering Green Fluorescent Protein as a Dual Functional TAG
Authors: Parambam, Rosanto I.
Advisor: Su, Wei-Wen
Issue Date: Dec 2002
Publisher: University of Hawaii at Manoa
Abstract: A novel dual functional green fluorescent protein (GFP) tag useful for monitoring and purifying a recombinant protein fused to the tag has been developed. A poly-histidine (6xHis) tag was inserted into a solvent exposed loop of the ll-stranded B-barrel GFP structure. Two variants (GFP172 and GFP157) were made depending on the site of insertion of the 6xHis tag. We produced the variants in Escherischia coli and purified them using immobilized metal affinity chromatography (IMAC). These purified GFP variants retained 65-68% of the fluorescence compared to having the tag inserted at the C-terminus. On comparison of recovery from a buffer by IMAC, it was found that greater than 80% of the protein could be recovered. Effect of temperature on the expression of these variants showed that GFP172 could be produced in soluble form at both 37°C and 28°C whereas negligible amounts of soluble GFP157 was obtained. GFP172 was fused to the C-terminus of Maltose Binding Protein (MBP) and the fusion protein was purified from E. coli lysate as well as from spiked tobacco leaf extracts. The primary advantage of this new GFP molecule is that it allows maximum flexibility for protein fusion since both N- and C-terminal ends are available for linking to a protein of interest. In conjunction with appropriate targeting/retention signals, this GFP tag can improve fusion protein accumulation and stability and at the same time perform the dual functions of protein monitoring and affinity purification. Preliminary studies were done to study the effect of a C-terminal HDEL sequence on the expression of granulocyte macrophage-colony stimulating factor (GM-CSF) fusion with GFP in tobacco suspension cultures. It was found that HDEL influences the stability of the fusion protein because in its absence, majority of the protein was not fluorescent.
Description: x, 110 leaves
URI/DOI: http://hdl.handle.net/10125/6955
Rights: All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Appears in Collections:M.S. - Molecular Biosciences and Bioengineering



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