Biosynthesis of human β2-adrenergic receptor in methylotrophic yeast Pichia pastoris and its purification

Abstract

Human β2-adrenergic receptor is among the most studied members of the family of G-protein coupled receptors. This receptor plays an important role in the work of vegetative nervous system and is a target for drugs used in heart and lung diseases. The crystal structure of the β2-adrenergic receptor was determined; however, its physiological role and molecular mechanisms of action are still not completely understood. In the present work, the pVR2ADRH vector which contains the gene encoding human β2-adrenergic receptor with a polyhistidine label at the C-terminus was obtained. The Pichia pastoris methylotrophic yeast GS115 strain cells were transformed by this vector. The yield of human β2-adrenergic receptor as a result of the heterologous expression in the P. pastoris cells was 20 mg/L of the culture. The β2-adrenergic receptor was isolated from the membrane fraction of yeasts and purified by means of a metal chelate affine and ion-exchange chromatography. Active β2-adrenergic receptor was isolated using a chromatography on the CL-4B alprenololsepharose. The final yield of the purified β2-adrenergic receptor was approximately 1 mg/L of the culture. A homogeneity of the β2-adrenergic receptor preparation was confirmed by a method of dynamic light scattering of a protein micellar solution.