Expression systems for bovine rhodopsin: a review of the progress made in the Khorana laboratory

Abstract

Here I will review the development of gene expression systems for production of bovine rhodopsin in the Khorana laboratory with particular focus on stable mammalian cell lines made using human embryonic kidney cells (HEK293S). The synthesis of a gene encoding bovine rhodopsin was completed in 1986. This gene was expertly designed with the built-in capacity for DNA duplex cassette replacement mutagenesis which made site-directed mutagenesis relatively straightforward. Intense effort was expended over several years in order to identify a gene expression system capable of producing rhodopsin in milligram amounts as required for biophysical studies. Mammalian expression systems, both transient and stable, were found to be the most favourable based on several criteria including receptor expression levels, correct folding and post translational processing, and capacity for purification of fully functional receptor. Transient expression using COS-1 cells was preferred for routine small-scale production of rhodopsin mutants, while HEK293S stable cell lines were used when milligram amounts of rhodopsin mutants were needed; for example, when conducting NMR studies.

Keywords: G protein-coupled receptors; Glycosylation; Membrane proteins; Sodium butyrate; Tetracycline-inducible.

Fig. 1

Expression vector pACHrhoC for construction of mammalian stable cell lines. The rhodopsin gene is controlled by the strong constitutively active CMV promoter. This plasmid also contains the gene for G418 resistance which is under control of the weak H₂L^d promoter. Integration of this plasmid into transcriptionally active regions of the chromosome is selected by growth of transfected HEK293S cells in the presence of high concentrations (2–3 mg/ml) of G418. Further details for construction and use of this plasmid are described in the original manuscript (Reeves et al. 1996). Copyright (1996) National Academy of Sciences, USA

Fig. 2

Immunoaffinity purification of a milligram of rhodopsin from a HEK293S stable cell line grown in suspension culture. A cell line stably transfected with pACHRhoC (Fig. 1) was grown in suspension culture (550 ml). Rhodopsin was purified from harvested cells by a single-step immunoaffinity procedure using Rho-1D4 Sepharose. Fractions were collected and analysed during the entire procedure. After loading the column (load) the column was washed with PBS (E1), a low salt buffer (E2), the same low salt buffer containing the 9mer elution peptide (E3) and finally PBS containing the 9mer elution peptide (E4). Correctly folded rhodopsin (A_280nm/A_500nm ratio of ~ 1.65) elutes sharply from this column from fractions collected using buffer E3. SDS-PAGE analysis followed by silver staining (A) and Western blot (B) is shown as an inset. Full details of this experiment are described in Reeves et al. (1996) from which this figure was reproduced. Copyright (1996) National Academy of Sciences, USA

Fig. 3

Expression vector pACMV-tetO-Rho for construction of inducible mammalian stable cell lines. This expression plasmid carries the bovine rhodopsin gene under the control of a human CMV promoter containing two tandem tetO DNA sequences. When this plasmid is integrated into the genome of HEK293S cells expressing the tetracycline repressor protein (TetR), expression from this hybrid CMV promoter is blocked. Stable cell lines transfected with this plasmid are obtained by using G418 selection. Optimal inducible expression from the CMV-tetO promoter is brought about by addition of tetracycline and sodium butyrate to the growth medium followed by further incubation for 60–72 h. Details for construction of this plasmid have been described previously (Reeves et al. 2002b) and this figure is reproduced from Fig. 1 in that publication. Copyright (2002) National Academy of Sciences, USA

Fig. 4

Isolation of a HEK293S cell line for production of glycoproteins containing short homogeneous N-glycans. HEK293S cells were subjected to chemical mutagenesis followed by growth in the presence of ricin to select for cell lines defective for complex N-glycan synthesis. Surviving HEK293S cell lines resistant to ricin were expanded and transfected with pMT4-Rho. Rhodopsin produced was purified and examined by SDS-PAGE followed by silver staining. Lanes 8 and 9 contain the control HEK293S and bovine retina rhodopsin, respectively. Rhodopsin purified from ricin-resistant HEK293S cell line candidate 15 (lane 7) no longer has a high molecular weight trailing smear but instead migrates like rhodopsin purified from bovine retinas. This image is from Reeves et al. (2002a). Copyright (2002) National Academy of Sciences, USA