[Biosynthesis and purification of human beta2-adrenergic receptor expressed in methylotrophic yeast Pichia pastoris].
Human beta2-adrenergic receptor is one of the most studied G-protein-coupled receptors. It plays a key role in autonomic nervous system and is a drug target in cardiovascular and pulmonary diseases. Despite the fact that its crystal structure was revealed, a physiological role and molecular mechanisms of its action remain largely unknown. We designed the construct pVR2ADRH, which contained the gene for human beta2-adrenergic receptor with a polyhistidine tag C-terminal extension. The recombinant DNA was used for transformation of the GS115 strain of Pichia pastoris. The heterologous expression level obtained was about 20 mg/l. The receptor was extracted from membrane fraction and was purified by metal-affinity and ion-exchange chromatography. The active receptors were isolated by alprenolol-sepharose CL-4B. The resulting level of purified human beta2-adrenergic receptor was approximately 1 mg per liter of culture. The homogeneity of the protein sample was confirmed by a dynamical light scattering analysis of the receptor's micellar solution.
A S
AS
Gerasimov
O A
OA
Zeĭnalov
M A
MA
Él'darov
A A
AA
shul'ga
D015870:Gene Expression; D006801:Humans; D010843:Pichia; D018343:Receptors, Adrenergic, beta-2 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins