title: Engineering, expression, purification, and physicochemical characterization of a chimeric protein, full-length cytochrome b(5)--green fluorescence protein (HMWb5--EGFP).
abstract: 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 HMWb(5)-EGFP consisting of full-length cytochrome b(5) (HMWb(5)) 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 HMWb(5)-EGFP was purified from cell membranes by using metal-affinity chromatography. It possesses physicochemical, spectral, and fluorescence properties of cytochrome b(5) 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 HMWb(5)-EGFP between the fluorophore of EGFP and heme of cytochrome b(5), and the distance between chromophores in the chimeric protein is approximately 67.3 A. The chimeric protein was shown to exist as a monomer in aqueous solution in the presence of detergents. The data indicate that the HMWb(5)-EGFP designed in the present work is a very promising model for modern biosensors and an instrument to study protein-protein interactions.
authors.affiliation: Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk, 220141, Belarus.
authors.forename: A V
authors.initials: AV
authors.lastname: Yantsevich
authors.forename: I N
authors.initials: IN
authors.lastname: Harnostai
authors.forename: O P
authors.initials: OP
authors.lastname: Lukashevich
authors.forename: A A
authors.initials: AA
authors.lastname: Gilep
authors.forename: S A
authors.initials: SA
authors.lastname: Usanov
mesh_terms: D002846:Chromatography, Affinity; D015786:Cytochromes b5 / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015786:Cytochromes b5