Structure and function in rhodopsin: a tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants

Abstract

Tetracycline-inducible HEK293S stable cell lines have been prepared that express high levels (up to 10 mg/liter) of WT opsin and its mutants only in response to the addition of tetracycline and sodium butyrate. The cell lines were prepared by stable transfection of HEK293S-TetR cells with expression plasmids that contained the opsin gene downstream of a cytomegalovirus promoter containing tetO sequences as well as the neomycin resistance gene under control of the weak H(2)L(d) promoter. The inducible system is particularly suited for overcoming problems with toxicity either due to the addition of toxic compounds, for example, tunicamycin, to the growth medium or due to the expressed protein products. By optimization of cell growth conditions in a bioreactor, WT opsin, a constitutively active opsin mutant, E113Q/E134Q/M257Y, presumed to be toxic to the cells, and nonglycosylated WT opsin obtained by growth in the presence of tunicamycin have been prepared in amounts of several milligrams per liter of culture medium.

Figure 1

Preparation of the vector pACMV-tetO-Rho (IV) for tetracycline-inducible opsin expression. The detailed procedure is in Materials and Methods. In brief, the construction involved four main steps, A–D. In A, the tetO operator sequences were inserted into the plasmid pCEP4(I) and the fragment 1 containing the CMV(tetO) promoter was isolated. In B, fragment 2 was prepared from the plasmid pACHEnc(II) by removal of CMV + Ach(nc). In C, fragments 1 and 2 were ligated to give the plasmid pACMV-tetO (IV). In D, the opsin gene isolated from the plasmid pMT4 was inserted into the plasmid pACMV-tetO(III) to give the vector pACMV-tetO-Rho. The multiple cloning site (MCS) located downstream of the CMV-tetO promoter in pACMV-tetO contains [(5′-3′) KpnI, PvuII, NheI, HindIII, NheI, NotI, XhoI, SfiI, BamHI].

Figure 2

Expression of the opsin gene under different conditions after growth of a stable cell line. Cells were grown in dishes to near confluence and then incubated for the time period shown after addition of fresh growth medium supplemented as indicated (a–d). Each bar shows the average amount of opsin produced by cells from duplicate culture dishes as measured by constituted rhodopsin (UV-visible difference spectroscopy; see Materials and Methods).

Figure 3

Growth of a cell line inducible for WT opsin expression in 1.1-liter suspension culture by using a bioreactor. The cell line was grown in the bioreactor as described in Materials and Methods. (A) Viable count calculated by counting cells samples removed from the bioreactor at 24-h intervals. (Error bars represent average of two counts from same time point; Materials and Methods.) The culture was supplemented with the growth medium and opsin expression was induced as indicated by arrows. (B) Samples collected throughout growth were solubilized and proteins were separated by SDS/PAGE (10%). Whole-cell proteins were detected by Coomassie blue staining. For immunodetection, the proteins were transferred from gels to nitrocellulose by electroblotting and opsin was visualized by immunodetection with anti-rhodopsin monoclonal antibody rho-1D4. Sup., supplementation; Ind., induction.

Figure 4

Effect of tunicamycin concentration on extent of opsin N-glycosylation. The tetracycline-inducible cell line producing WT opsin was grown to near confluence in 10-cm-diameter cell culture dishes. Spent medium was removed and replaced with fresh medium containing different concentrations (0–2.5 μg/ml) of tunicamycin. Three hours later, expression of opsin was induced by further supplementation of the growth medium with tetracycline and sodium butyrate. Cells were harvested 48 h later and treated with 11-cis-retinal to constitute the rhodopsin pigment. Rhodopsin was purified and examined (0.5 μg) by SDS/PAGE with silver stain for detection. Rhodopsin purified from bovine rod outer segments was loaded in lane 2, whereas molecular weight protein standards (M) were in lanes 1 and 8.

Figure 5

Purification of nonglycosylated rhodopsin from a portion of the cells grown in a 5.5-liter bioreactor by rho-1D4 immunoaffinity chromatography. Elution buffers were: E1, buffer H; E2, buffer I; E3, buffer J; E4, buffer K. Absorption at 280 nm (A₂₈₀) (filled circles) and at 500 nm (A₅₀₀) (open circles) was recorded as indicated. (Inset) SDS/PAGE (10% gel) examination of selected fractions eluted from the column as visualized by silver stain and immunoblot after electroblotting to nitrocellulose. M, molecular weight standards; S, solubilized cell extract; F, flowthrough containing proteins that do not bind to rho-1D4-Sepharose.

Figure 6

Purification by rho-1D4 immunoaffinity chromatography of rhodopsin mutant, E113Q/E134Q/M257Y, from a portion of the cells grown in a 1.1-liter bioreactor culture. Elution buffers were: E1, buffer C; E2, buffer D; E3, buffer E; E4, buffer F. Absorption at 280 nm (A₂₈₀) (filled circles) and 380 nm (A₃₈₀) (open circles) was recorded as indicated. (Inset) SDS/PAGE (10% gel) examination of selected fractions eluted from the column as visualized by silver stain and immunoblot after electroblotting to nitrocellulose. M, molecular weight standards; S, solubilized cell extract; F, flowthrough containing proteins that do not bind to rho-1D4-Sepharose.