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

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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

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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.

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KEY WORDS: microsomal cytochrome b₅, green fluorescence protein (EGFP) from Aequorea, expression in Escherichia coli, affinity chromatography, purification

DOI: 10.1134/S0006297907010099

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