Production of Olfactory Receptors Using Commercial E. coli Cell-free Systems - Bioelectronic Nose: Integration of Biotechnology and Nanotechnology - page 111

Chapter 7
Production of Olfactory Receptors Using
Commercial E. coli Cell-free Systems

Karolina Corin, Xiaoqiang Wang and Shuguang Zhang

Abstract The first bottleneck in olfactory receptor (OR) structural and functional
studies is to produce sufficient quantities of soluble, functional, and stable recep-
tors. Other production systems have been used and summarized in other chapters
of this topic. We here show that commercial cell-free in vitro translation systems
can be used to produce milligrams of soluble and functional olfactory receptors
within several hours directly from plasmid DNA with select optimal detergents.
These olfactory receptors can be purified using immunoaffinity 1D4 monoclonal
antibody rhodopsin-tag and gel filtration, and can be analyzed using gel electropho-
resis and with other standard techniques. The olfactory receptors and other scent-
related receptors produced by the cell-free method fold properly and are able to bind
their odorants.

7.1 Production of Olfactory Receptors is Required for

Study and New Technology

The molecular basis of olfaction is poorly understood, primarily due to the extreme
difficulty of producing sufficient quantities of soluble and functional olfactory re-
ceptors (ORs). Olfactory receptors belong to the G protein-coupled receptor family,
which is characterized by seven transmembrane helical segments arranged in a bar-
rel-like conformation. These transmembrane regions, which include the receptor-
binding pocket, can cause problems with protein expression, and make it difficult to
functionally stabilize the receptors outside of their native membrane environment.
Receptor production in eukaryotic or bacterial cells frequently encounters problems
such as low yields, cell toxicity, protein degradation, protein inhomogeneity and
aggregation in internal compartments or inclusion bodies [ 1 - 5 ]. Cell-free in vitro
translation is an alternative and enabling method allowing for rapid, cost-effective,
high-yield protein expression [ 6 - 11 ]. The produced olfactory receptor proteins can