Chapter 3: Baculoviral Expression of an Integral Membrane Protein for Structural Studies
Dean R. Madden & Markus Safferling
The baculovirus system has proven successful for the expression of integral membrane proteins for structural studies. A recombinant baculovirus, in which the gene of interest is placed under the control of the late-stage polyhedrin promoter, serves as the starting point for viral expansion and protein expression studies. Using large-scale insect cell culture techniques together with a filter-binding assay for protein function, the conditions of expression, purification, and solubilization can be optimized. As applied to the glutamate receptor ion channel subunit GluR2, this approach yields milligram quantities of pure, active protein, which have been used for single-particle electron microscopic analysis of the receptor structure. Detergent exchange protocols are also discussed, as a prerequisite for two-dimensional crystallization trials.