[Biosynthesis and purification of human beta2-adrenergic receptor expressed in methylotrophic yeast Pichia pastoris]

Mol Biol (Mosk). 2012 Mar-Apr;46(2):308-16.
[Article in Russian]

Abstract

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.

Publication types

  • English Abstract

MeSH terms

  • Gene Expression*
  • Humans
  • Pichia
  • Receptors, Adrenergic, beta-2* / biosynthesis
  • Receptors, Adrenergic, beta-2* / chemistry
  • Receptors, Adrenergic, beta-2* / genetics
  • Receptors, Adrenergic, beta-2* / isolation & purification
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification

Substances

  • Receptors, Adrenergic, beta-2
  • Recombinant Proteins