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Engineering, Expression, Purification, and Physicochemical Characterization of a Chimeric Protein, Full-Length Cytochrome b5-Green Fluorescence Protein (HMWb5-EGFP)


A. V. Yantsevich, I. N. Harnostai, O. P. Lukashevich, A. A. Gilep, and S. A. Usanov*

Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, ul. Kuprevicha 5, 220141 Minsk, Belarus; fax: 375 (172) 637-274; E-mail: usanov@iboch.bas-net.by

* To whom correspondence should be addressed.

Received July 10, 2006; Revision received September 11, 2006
In this article we report on construction of expression vector, heterologous expression in Escherichia coli, isolation, purification, and physicochemical characterization of an artificial chimeric protein HMWb5-EGFP consisting of full-length cytochrome b5 (HMWb5) and green fluorescence protein (EGFP) from Aequorea. Optimization of expression conditions yielded an expression level up to 1500 nmol of chimeric protein per liter of culture. Recombinant chimeric protein HMWb5-EGFP was purified from cell membranes by using metal-affinity chromatography. It possesses physicochemical, spectral, and fluorescence properties of cytochrome b5 and EGFP indicating independent character of protein folding in frames of the chimera. It is shown that there is a fluorescent resonance energy transfer in HMWb5-EGFP between the fluorophore of EGFP and heme of cytochrome b5, and the distance between chromophores in the chimeric protein is approximately 67.3 Å. The chimeric protein was shown to exist as a monomer in aqueous solution in the presence of detergents. The data indicate that the HMWb5-EGFP designed in the present work is a very promising model for modern biosensors and an instrument to study protein-protein interactions.
KEY WORDS: microsomal cytochrome b5, green fluorescence protein (EGFP) from Aequorea, expression in Escherichia coli, affinity chromatography, purification

DOI: 10.1134/S0006297907010099