Large-scale production and purification of functional recombinant bovine rhodopsin with the use of the baculovirus expression system

Abstract

Here we describe a generic procedure for the expression and purification of milligram quantities of functional recombinant eukaryotic integral membrane proteins, exemplified by hexahistidine-tagged bovine rhodopsin. These quantities were obtained with the recombinant baculovirus/Sf9 insect cell-based expression system in large-scale bioreactor cultures with the use of a serum-free and protein-free growth medium. After optimization procedures, expression levels up to 4 mg/l were established. The recombinant rhodopsin could be purified with high overall yield by using immobilized-metal-affinity chromatography on Ni(2+)-agarose. After reconstitution into a native lipid environment, the purified protein was functionally indistinguishable from native rhodopsin with regard to the following parameters: spectral absorbance band, structural changes after photoactivation, and G-protein activation. The procedures developed can be adapted to other membrane proteins. The ability to produce and purify tens of milligrams of functional recombinant eukaryotic membrane protein meets the ever-increasing demand of material necessary to perform detailed biochemical and structural biophysical studies that are essential in unravelling their working mechanism at a molecular level.

Large‐scale overproduction, functional purification and ligand affinities of the His‐tagged human histamine H1 receptor

“…Biophysical studies that can provide detailed structural and functional information require mg amounts of purified protein. There are several reports claiming a high level production of functional GPCRs in insect cells in the range of 2–4 mg·mL −1 after infection with the corresponding recombinant baculovirus [19,23–25,33,40,41]. Recent advances include further development of the system for production of multisubunit protein complexes and coexpression of protein‐modifying enzymes to improve heterologous protein production [16].…”

“…Removal of the sucrose in the H1 receptor proteoliposome fraction by dilution with five volumes of Milli‐Q water, and subsequent centrifugation for 30 min (80 000 g at 4 °C) yielded a visible precipitate, which was subsequently stored as a pellet at −80 °C for future studies. Protein was determined using the Bradford assay (Bio‐Rad, Melville, NY, USA) according to the manufacturer's instructions using bovine rhodopsin for calibration [19].…”

“…Hence we decided to select the H1 receptor for large‐scale production and purification studies. For rhodopsin, good production levels were obtained in a protein‐free medium (Insect Xpress) [19]. Therefore we compared H1 receptor production levels in Insect Xpress and in the established standard serum‐supplemented TNM‐FH medium.…”

“…This probably is due to some cross‐reactivity of the His‐tag antibody with endogenous proteins as well as to the presence of misfolded or otherwise nonfunctional receptor [27]. It has been previously reported that the MOI and the time‐point of infection in the cellular growth cycle are important parameters in determining volumetric production levels and optimal time of harvesting [19]. Final levels did not vary significantly when cells were infected in their early mid‐exponential growth phase for an MOI of 0.1, 1 and 10, but usually lagged behind at a MOI of 0.01.…”

“…To obtain sufficient amounts of purified H1 receptor for structural and mechanistic studies by state‐of‐the‐art biophysical techniques, we have designed procedures for overproduction, purification and reconstitution. We exploit the baculovirus/insect cell system, that has been successfully used for overproduction of a variety of GPCRs in Sf9 cells [16–21], an ovarian cell line derived from Spodoptera frugiperda , which is able to perform most eukaryotic post‐translational modifications, such as phosphorylation, fatty acid acylation, disulfide bond formation and glycosylation [22–25]. The insect cell system has the advantage that large‐scale suspension cultures can be grown using commercially available protein‐free media.…”