Heterologous Expression of Bovine Opsin in Pichia Pastoris

Published

March 1, 2000

Author(s)

N G. Abdulaev, K D. Ridge

Abstract

The overproduction of bovine opsin and structurally related receptors in a variety of heterologous systems has so far proven quite difficult and consequently limited both biophysical and structural investigations. Therefore, a highly efficient expression system capable of producing modified opsin or its defined domains in the quantities required for these analyses would be beneficial. Recombinant opsin has been produced by transient and/or stable transfection of COS-1 and human embryonic kidney (HEK) cells,^1-2 RNA injection of Xenopus laevis oocytes,⁴ in baculovirus infected Sf9 insect cells,⁵ by RNA translation in wheat germ extracts,⁶ and recently, in Saccharomyces cerevisiae.⁷ Although expression in COS-1 and HEK293 cells remain the methods of choice for most structure-function studies on rhodopsin, their use for large-scale production is rather laborious and expensive. The purpose of this chapter is to describe methods for the functional expression of bovine opsin in Pichia pastoris,⁸ a methylotrophic yeast capable of producing considerable quantities of foreign protein.⁹ The focus is on the construction of a suitable expression vector, conditions for stable transformation and expression in P. pastoris cells, and the purification and characterization of yeast expressed rhodopsin.

Citation

Heterologous Expression of Bovine Opsin in Pichia Pastoris

Volume

315

Issue

Chap. 1

Publisher Info

Methods in Enzymology Chapter,

Pub Type

Books

Keywords

membrane protein, protein folding, receptor, signal transduction, vision, yeast

Citation

Abdulaev, N. and Ridge, K. (2000), Heterologous Expression of Bovine Opsin in Pichia Pastoris, Methods in Enzymology Chapter, (Accessed April 25, 2024)