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• W2103208001 abstract "For rapid single-step purification of recombinant rhodopsin, a baculovirus expression vector was constructed containing the bovine opsin coding sequence extended at the 3′-end by a short sequence encoding six histidine residues. Recombinant baculovirus-infected Spodoptera frugiperda cells produce bovine opsin carrying a C-terminal histidine tag (v-opshis6x). The presence of this tag was confirmed by immunoblot analysis. Incubation with 11-cis-retinal produced a photosensitive pigment (v-Rhohis6x) at a level of 15–20 pmol/106 cells. The histidine tag was exploited to purify v-Rhohis6x via immobilized metal affinity chromatography. Optimized immobilized metal affinity chromatography yielded a binding capacity of ≥35 nmol of v-Rhohis6x per ml of resin and purification factors up to 500. Best samples were at least 85% pure, with an average purity of 70% (A280 nm/α500 nm = 2.5 ± 0.4, n = 7). Remaining contamination was largely removed upon reconstitution into lipids, yielding rhodopsin proteoliposomes with a purity over 95%.Spectral analysis of v-Rhohis6x showed a small but significant red shift (501 ± 1 nm) compared to wild type rhodopsin (498 ± 1 nm). The pKa of the Meta I ↔ Meta II equilibrium in v-Rhohis6x is down-shifted from 7.3 to 6.4 resulting in a significant shift at pH 6.5 toward the Meta I photointermediate. Both effects are reversed upon increasing the ionic strength. FT-IR analysis of the Rho → Meta II transition shows that the corresponding structural changes are identical in wild type and v-Rhohis6x. For rapid single-step purification of recombinant rhodopsin, a baculovirus expression vector was constructed containing the bovine opsin coding sequence extended at the 3′-end by a short sequence encoding six histidine residues. Recombinant baculovirus-infected Spodoptera frugiperda cells produce bovine opsin carrying a C-terminal histidine tag (v-opshis6x). The presence of this tag was confirmed by immunoblot analysis. Incubation with 11-cis-retinal produced a photosensitive pigment (v-Rhohis6x) at a level of 15–20 pmol/106 cells. The histidine tag was exploited to purify v-Rhohis6x via immobilized metal affinity chromatography. Optimized immobilized metal affinity chromatography yielded a binding capacity of ≥35 nmol of v-Rhohis6x per ml of resin and purification factors up to 500. Best samples were at least 85% pure, with an average purity of 70% (A280 nm/α500 nm = 2.5 ± 0.4, n = 7). Remaining contamination was largely removed upon reconstitution into lipids, yielding rhodopsin proteoliposomes with a purity over 95%. Spectral analysis of v-Rhohis6x showed a small but significant red shift (501 ± 1 nm) compared to wild type rhodopsin (498 ± 1 nm). The pKa of the Meta I ↔ Meta II equilibrium in v-Rhohis6x is down-shifted from 7.3 to 6.4 resulting in a significant shift at pH 6.5 toward the Meta I photointermediate. Both effects are reversed upon increasing the ionic strength. FT-IR analysis of the Rho → Meta II transition shows that the corresponding structural changes are identical in wild type and v-Rhohis6x. Rhodopsin is the major component of the outer segments of the vertebrate rod photoreceptor cell. This visual pigment consists of an integral membrane protein to which a chromophore, 11-cis-retinal, is covalently linked via a protonated Schiff base. Rhodopsin triggers the conversion of photon energy (light) into a graded membrane potential. The absorption of a photon leads to a number of discrete conformational changes in the protein moiety of the pigment (sequel of photointermediates → photocascade), finally resulting in the exposure of G-protein binding sites at the cytoplasmic surface of the protein. In the past decade, research has focused on analyzing the relationship between the structure of the receptor and its functional properties. Heterologous expression of the protein in combination with site-specific mutagenesis has become an attractive way to study this relationship. Several expression systems capable of in vitro biosynthesis of opsin have been described (1Oprian D.D. Molday R.S. Kaufman R.J. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8874-8878Crossref PubMed Scopus (387) Google Scholar, 2Janssen J.J.M. VanDeVen W.J.M. VanGroningen-Luyben W.A.H.M. Roosien J. Vlak J.M. DeGrip W.J. Mol. Biol. Rep. 1988; 13: 65-71Crossref PubMed Scopus (27) Google Scholar, 3Khorana H.G. Knox B.E. Nasi E. Swanson R. Thompson D.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7917-7921Crossref PubMed Scopus (82) Google Scholar, 4Nathans J. Weitz C.J. Agarwal N. Nir I. Papermaster D.S. Vision Res. 1989; 29: 907-914Crossref PubMed Scopus (42) Google Scholar, 5Zozulya S.A. Gurevich V.V. Zvyaga T.A. Shirokova E.P. Dumler I.L. Garnovskaya M.N. Natochin M.Y. Shmukler B.E. Badalov P.R. Protein Eng. 1990; 3: 453-458Crossref PubMed Scopus (17) Google Scholar). Expression levels in these systems are usually quite low compared to total cell protein (<0.5%) and even to total membrane protein. For most analyses, recombinant rhodopsin therefore has to be extensively purified. Several methods have been described for the purification of recombinant rhodopsin (1Oprian D.D. Molday R.S. Kaufman R.J. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8874-8878Crossref PubMed Scopus (387) Google Scholar, 4Nathans J. Weitz C.J. Agarwal N. Nir I. Papermaster D.S. Vision Res. 1989; 29: 907-914Crossref PubMed Scopus (42) Google Scholar, 6DeCaluwé G.L.J. VanOostrum J. Janssen J.J.M. DeGrip W.J. Methods Neurosci. 1993; 15: 307-321Crossref Scopus (22) Google Scholar). These methods often have the disadvantage that the obtained samples are still contaminated to a various extent with proteins derived from the cells, used for recombinant protein production (4Nathans J. Weitz C.J. Agarwal N. Nir I. Papermaster D.S. Vision Res. 1989; 29: 907-914Crossref PubMed Scopus (42) Google Scholar, 6DeCaluwé G.L.J. VanOostrum J. Janssen J.J.M. DeGrip W.J. Methods Neurosci. 1993; 15: 307-321Crossref Scopus (22) Google Scholar). Quite pure preparations can be obtained using immunoaffinity chromatography (1Oprian D.D. Molday R.S. Kaufman R.J. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8874-8878Crossref PubMed Scopus (387) Google Scholar). However, this approach is expensive (monoclonal antibody, peptides for elution), laborious (antibody production, purification, and coupling), and fairly inefficient (low column capacity, recovery only in the order of 50%). Hence, this procedure is not very suitable for production of larger amounts (1–10 mg of purified protein) required for structural studies (crystallization, FT-IR spectroscopy, 1The abbreviations used are: FT-IRFourier transform infraredIMACimmobilized metal affinity chromatographyv-Rhowild-type regenerated opsin produced in vitro by recombinant baculovirusv-Rhohisg6xC-terminally histidine-tagged regenerated opsin produced in vitro by recombinant baculovirusDoMdodecyl-β-1-maltosidePIPES1,4-piperazinediethanesulfonic acid;MES,4-morpholineethanesulfonic acidHEPPS4-(2-hydroxyethy1)-1-piperazinepropanesulfonic aciddsdouble-strandedNTAnitrilotriacetic acidPAGEpolyacrylamide gel electrophoresis NMR spectroscopy). Fourier transform infrared immobilized metal affinity chromatography wild-type regenerated opsin produced in vitro by recombinant baculovirus C-terminally histidine-tagged regenerated opsin produced in vitro by recombinant baculovirus dodecyl-β-1-maltoside 1,4-piperazinediethanesulfonic acid;MES,4-morpholineethanesulfonic acid 4-(2-hydroxyethy1)-1-piperazinepropanesulfonic acid double-stranded nitrilotriacetic acid polyacrylamide gel electrophoresis The recombinant baculovirus-based expression system is an excellent system for the production of larger amounts of recombinant bovine rhodopsin (6DeCaluwé G.L.J. VanOostrum J. Janssen J.J.M. DeGrip W.J. Methods Neurosci. 1993; 15: 307-321Crossref Scopus (22) Google Scholar, 7Janssen J.J.M. DeCaluwé G.L.J. DeGrip W.J. FEBS Lett. 1990; 260: 113-118Crossref PubMed Scopus (19) Google Scholar). Thus far, we have used concanavalin A-Sepharose affinity chromatography to purify rhodopsin produced in this system (6DeCaluwé G.L.J. VanOostrum J. Janssen J.J.M. DeGrip W.J. Methods Neurosci. 1993; 15: 307-321Crossref Scopus (22) Google Scholar), which, combined with reconstitution into proteoliposomes, yields reasonably pure preparations (60–80%). However, it is quite inefficient. Because of contaminating viral glycoproteins, the column capacity for recombinant rhodopsin is small, and a laborious elution profile has to be applied. Here we report on an alternative approach, using a histidine tag engineered onto the C terminus of bovine opsin. Histidine tagging for purification of recombinant proteins by means of IMAC has been used for a number of proteins both in prokaryotic (8Gentz R. Certa U. Takaes B. Matile H. Döbeli H. Pink R. Mackay M. Bone N. Scafi J.G. EMBO J. 1988; 7: 225-230Crossref PubMed Scopus (42) Google Scholar, 9Hochuli E. Bannwarth W. Dbbeli H. Gentz R. Stüber D. Bio/Technology. 1988; 6: 1321-1325Crossref Scopus (964) Google Scholar, 10Smith M.C. Furman T.C. Ingolia T.D. Pidgeon C. J. Biol. Chem. 1988; 263: 7211-7215Abstract Full Text PDF PubMed Google Scholar) and eukaryotic (11Janknecht R. DeMartynoff G. Lou J. Hipskind R.A. Nordheim A. Stunnenberg H.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8972-8976Crossref PubMed Scopus (414) Google Scholar) expression systems, including recombinant baculovirus (12Chen X. Brash A.R. Funk C.D. Eur. J. Biochem. 1993; 214: 845-852Crossref PubMed Scopus (64) Google Scholar, 13Wang M. Bentley W.E. Vakharia V. Biotechnol. Bioeng. 1994; 43: 349-356Crossref PubMed Scopus (25) Google Scholar, 14Reddy R.G. Yoshimoto T. Yamamoto S. Funk C.D. Marnett L.J. Arch. Biochem. Biophys. 1994; 312: 219-226Crossref PubMed Scopus (23) Google Scholar). None of these proteins, however, belonged to the superfamily of heptahelical G-protein-coupled membrane receptors of which rhodopsin is a member. Membrane proteins have to be solubilized with the help of specific detergents which may impair affinity techniques. Hence, we have evaluated IMAC for purification of recombinant rhodopsin. Here we will demonstrate that an adapted IMAC allows relatively simple, highly efficient single-step purification of histidine-tagged rhodopsin with an excellent purification factor (≥500). The procedure we developed is directly applicable to other membrane (receptor) proteins. Interestingly, the C-terminally located histidine tag slightly influences spectral properties (3 nm red-shift) and photocascade (downshift of the pKa of the Meta I ↔ Meta II equilibrium) of rhodopsin, however, without perturbing the structural changes accompanying the photocascade. The effects are fully reversible at higher ionic strength and probably represent unexpected electrostatic effects on selected rhodopsin properties. Materials—Dodecyl-β-1-maltoside (DoM) and nonyl-β-1-glucoside were prepared as described previously (15DeGrip W.J. Bovee-Geurts P.H.M. Chem. Phys. Lipids. 1979; 23: 321-335Crossref Scopus (139) Google Scholar). PIPES, HEPPS and MES buffers were purchased from Research Organics Inc., Trolox from Aldrich, and leupeptin from Sigma. AgCl windows were obtained from Fisher Scientific Co. Construction of the Histidine Tag Transfer Vector (pAcJAC2)—A unique BamHI restriction sequence was engineered in front of the stop codon at the 3′-end of the cDNA-encoding bovine opsin (16Nathans J. Hogness D.S. Cell. 1983; 34: 807-814Abstract Full Text PDF PubMed Scopus (482) Google Scholar) using the Bio-Rad MutaGene kit (Bio-Rad) according to the manufacturer’s instructions. This changed the original 3′-end coding sequence from 5′caa gtg gcg cct gcc taa gccc-3′ to 5′-caa gtg gat cct gcc taa gccc-3′ (Fig. 1B). Into this BamHI site we inserted a short synthetic dsDNA sequence with BamHI overhangs using the two complementary oligonucleotides 5′-gatcctcaccatcaccatcaccactagt-3′ and 5′-gatcactagtggtgatggtgatggtgag-3′. The resulting transfer vector, designated pAcJAC2 (Fig. 1A), encodes bovine opsin carrying 6 histidines at the C terminus (Fig. 1B). The sequence was confirmed by dideoxysequencing. We used pAcJAC2 to generate a recombinant baculovirus as described previously (17Janssen J.J.M. Mulder W.R. DeCaluwé G.L.J. Vlak J.M. DeGrip W.J. Biochim. Biophys. Acta. 1991; 1089: 68-76Crossref PubMed Scopus (27) Google Scholar). Regeneration and Purification of v-Rhohis6x—Baculovirus propagation, transfection, isolation of recombinant virus, and Sf9 insect cell culture are performed as described previously (17Janssen J.J.M. Mulder W.R. DeCaluwé G.L.J. Vlak J.M. DeGrip W.J. Biochim. Biophys. Acta. 1991; 1089: 68-76Crossref PubMed Scopus (27) Google Scholar, 18Summers M.D. Smith G.E. Texas Exp. Station Bull. 1987; 1555Google Scholar). Viral infection was performed at a multiplicity of infection of 5. Cells were harvested 3 days post-infection by centrifugation (4,000 × g, 10 min, 4 °C). Regeneration of v-opshis6x into v-Rhohis6x was accomplished in total cellular membrane preparations. Briefly, cell pellets were resuspended in buffer A (6.5 mm PIPES, 10 mm EDTA, 5 mm dithioerythritol, and 100 ng/ml leupeptin, pH 6.5) at 108 cells/ml and lysed upon homogenization (Potter-Elvehjem-tube). Homogenized cells were centrifuged (30,000 × g, 20 min, 4 °C), and the pellet was resuspended in buffer β (20 mm PIPES, 130 mm NaCl, 10 mm KCl, 3 mm MgCl2, 2 mm CaCl2, 0.1 mm EDTA, and 100 ng/ml leupeptin, pH 6.5). All subsequent manipulations were performed in a nitrogen atmosphere and under dim red light (Schott-Jena, RG 645 cut-off filter). Retina lipids were added in 100-fold molar excess over v-opshis6x, followed by 11-cis-retinal in a small volume of dimethylformamide (10-fold molar excess over v-opshis6x) and solid dodecyl-β-1-maltoside (DoM) to 0.5 mm. Samples were rotated for 2 h at room temperature and centrifuged (30,000 × g, 20 min, 4 °C). v-Rhohis6x was solubilized from the pellet in the same buffer that was used to bind v-Rhohis6x to the Ni2+ nitrilotriacetic acid agarose resin (Ni2+-NTA; Qiagen). Several buffers were evaluated for the IMAC. Buffer C consisted of 20 mm HEPPS, 0.5 M NaCl, 0.1 mm phenylmethylsulfonyl fluoride, 100 ng/ml leupeptin, 1 mm imidazole, 50 μμ Trolox, and 20 mm DoM (pH 8.0). Buffer D was identical with buffer C, except for 10 mm imidazole instead of 1 mm. Buffer E was identical with buffer C, except for buffer and pH: PIPES (pH 6.5) was used instead of HEPPS (8.0). The regenerated pellets were incubated with these buffers (1 ml/108 cells) for 2 h at room temperature to solubilize v-Rhohis6x. Extracts were centrifuged (30,000 × g, 20 min, 4 °C) to remove insoluble material, and glycerol was added to a concentration of 15% (w/v). The solubilized v-Rhohis6x was then applied to the Ni2+-NTA column at a flow rate of 1–2 ml/h for 16 h at 4 °C under continuous recycling. This flow rate was maintained throughout the entire procedure. Typically, we used a column volume of 1 ml for the v-Rhohis6x extract from 1–2 × 109 infected insect cells. Unbound material was eluted from the column with the same buffer that was used for loading of the sample except that it was supplemented with 15% (w/v) glycerol and 20 mm nonylglycose instead of 20 mm DoM (10 column volumes). This was followed by a second rinse using the same buffer, but now containing 25 mm imidazole (4 volumes). The bound v-Rhohis6x was then eluted from the column using the same buffer (C, D, or E) supplemented with 100 mm imidazole and containing 20 mm nonylglucose instead of DoM and 130 mm instead of 500 mm NaCl (6 volumes). Reconstitution of Purified v-Rhohis6x into Retina Lipids—Retina lipids were extracted from illuminated bovine retinae using standard procedures (19Hendriks T. Klompmakers A.A. Daemen F.J.M. Bonting S.L. Biochim. Biophys. Acta. 1976; 433: 271-281Crossref Scopus (46) Google Scholar). The lipids were stored at –80 °C in the dichloromethane/methanol extract. Their concentration was determined by a modified Fiske-Subbarow phosphate assay (20Broekhuyse R.M. Biochim. Biophys. Acta. 1968; 152: 307-315Crossref PubMed Scopus (289) Google Scholar). The aliquot required for reconstitution is dried with argon gas, dissolved in a small volume of methanol, and then diluted with buffer β containing 20 mm nonylglucose. Purified v-Rhohis6x was concentrated to 3–10 nmol/ml on an Omega 30K filter (Filtron, Northborough, MA) and mixed with 1 volume of retina lipid extract (100-fold molar excess). This mixture was layered on top of a sucrose step gradient (10%, 20%, and 45% (w/w) in 3 × diluted buffer B) and spun overnight (100,000 × g, ≥ 16 h, 4 °C). Reconstituted v-Rhohis6x was collected from the 20% and/or 45% interface. Collected proteoliposomes were either diluted with the required buffer, and used for analysis, or with distilled water, pelleted (80,000 × g, 1 h, 4 °C), and stored at –80 °C until further use. Immunoblot Analysis—Proteins were separated on a 12.5% SDS-polyacrylamide gel (21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), transferred to nitrocellulose (Schleicher and Schull BA85, pore size 85), and probed with monoclonal or polyclonal antibodies (17Janssen J.J.M. Mulder W.R. DeCaluwé G.L.J. Vlak J.M. DeGrip W.J. Biochim. Biophys. Acta. 1991; 1089: 68-76Crossref PubMed Scopus (27) Google Scholar). The anti-opsin antibodies Rho-1D4 (monoclonal) and CERNJS858 (polyclonal) have been described before (22Molday R.S. MacKenzie D. Biochemistry. 1983; 22: 653-660Crossref PubMed Scopus (355) Google Scholar, 23DeGrip W.J. Progr. Retinal Res. 1985; 4: 137-180Crossref Scopus (21) Google Scholar) and were used in 1:1000 dilution. The anti-histidine tag antiserum was obtained from Cappel, Organon Teknika N.V., Turnhout, Belgium and was also used 1:1000. UV/Vis Spectroscopy and Photolysis—UV/Vis spectroscopy to determine absorbance band shape and λmax was routinely performed at pH 6.5 in buffer β supplemented with 20 mm DoM using a Perkin-Elmer λ15 recording spectrophotometer. Detergent was added to prevent light-scattering artifacts. The spectrum of detergent-solubilized samples was recorded, after addition of 1 M hydroxylamine to 50 mm, before and after illumination (5 min, 300-watt light bulb, KG1 heat filter, Schott, Mainz, FRG). For accurate determination of the λmax, difference spectra were used (illuminated spectrum subtracted from the “dark” one). The late photointermediates Meta I, Meta II, and Meta III were studied at 10 °C in proteoliposomes of v-Rhohis6x, using isotonic solutions buffered with 20 mm PIPES (pH 6.5–7.0), 20 mm MES (pH 5.5–6.0), or 20 mm HEPPS (pH 7.5–9.5) (24Cotton R.G.H. Mutat. Res. 1993; 285: 125-144Crossref PubMed Scopus (241) Google Scholar). The photocascade was triggered by illumination for 15 s (Schott OG530 cut-off filter), and spectra were recorded every 3 minutes. After 30 min, hydroxylamine was added to 50 mm to convert all photoproducts into opsin and retinaloxime, and remaining rhodopsin was bleached away by a 5-min illumination. Fourier Transform Infrared (FT-IR) Difference Spectroscopy—FT-IR spectra of the rhodopsin → Meta II transition were recorded in principle as described previously (25Rothschild K.J. Gillespie J. DeGrip W.J. Biophys. J. 1987; 51: 345-350Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 26DeGrip W.J. Gray D. Gillespie J. Bovee-Geurts P.H.M. VanDenBerg E. Lugtenburg J. Rothschild K.J. Photochem. Photobiol. 1988; 48: 497-504Crossref PubMed Scopus (69) Google Scholar). Briefly, 1 nmol samples were deposited on AgCl windows by isopotential spin-drying (27DeGrip W.J. Gillespie J. Rothschild K.J. Biochim. Biophys. Acta. 1985; 809: 97-106Crossref PubMed Scopus (48) Google Scholar). The resulting films were hydrated with 3 μl of MES buffer (pH 5.5), covered with another AgCl window, sealed with Teflon tape, and mounted into a variable temperature cell (Specac P/N21500, Kent, UK). FT-IR spectra were recorded on a Mattson Cygnus 100 spectrometer (Madison, WI) equipped with a liquid nitrogen-cooled, narrow band MCT detector. Sample hydration was monitored by the ratio between the peak absorbance near 3400 cm–1 (OH stretching mode of water) and near 2900 cm–1 (CH stretching mode of protein and lipids). Rhodopsin → Meta II difference spectra were recorded at 10 °C. Spectra were taken at 8 cm–1 resolution in 5-min blocks before and after illumination at (1280 scans/spectrum). Each sample was illuminated for 30 s in the spectrometer under computer control using a 12-V 20-watt halogen lamp equipped with Schott KG1 and OG530 cut-off filters. Difference spectra were computed by subtracting spectra before illumination from spectra taken after illumination, using the EXPERT-IR software (Mattson). Construction of the Histidine Tag Transfer Vector pAcJAC2 and Generation of Recombinant Virus—In order to introduce a tag consisting of six histidines at the C terminus of bovine opsin, the transfer vector pAcJAC2 was constructed (Fig. 1A). pAcJAC2 is derived from the baculovirus transfer vector pAcDZ1 (28Zuidema D. Schouten A. Usmany M. Maule A.J. Belsham G.J. Roosien J. Klinge-Roode E.C. VanLent J.W.M. Vlak J.M. J. Gen. Virol. 1990; 71: 2201-2209Crossref PubMed Scopus (76) Google Scholar). A short synthetic sequence encoding a histidine tag followed by a stop codon was introduced at the C terminus using two complementary oligonucleotides (Fig. 1B). As a result, the two alanine residues at the C terminus of opsin (Ala-346 and Ala-348) were substituted by an aspartic acid and the first histidine residue, respectively (Fig. 1B). These substitutions concern residues, which are not highly conserved. Also, the aspartic acid residue introduced should partly counterbalance the additional charge carried by the histidine tag. In pAcJAC2, opsin biosynthesis is controlled by the polyhedrin promoter while the small heat shock promoter of hsp70, drives the biosynthesis of β-galactosidase, which functions as a reporter enzyme (17Janssen J.J.M. Mulder W.R. DeCaluwé G.L.J. Vlak J.M. DeGrip W.J. Biochim. Biophys. Acta. 1991; 1089: 68-76Crossref PubMed Scopus (27) Google Scholar, 28Zuidema D. Schouten A. Usmany M. Maule A.J. Belsham G.J. Roosien J. Klinge-Roode E.C. VanLent J.W.M. Vlak J.M. J. Gen. Virol. 1990; 71: 2201-2209Crossref PubMed Scopus (76) Google Scholar). Expression of v-opshis6x using Recombinant Virus AcNPV/opshis—Protein samples derived from recombinant virus-infected Sf9 cells were analyzed by immunoblot, using a polyclonal antiserum elicited against bovine opsin (Fig. 2A). Wild-type virus-infected Sf9 cells are used as a negative control, and native bovine opsin and wild-type v-ops as positive controls (Fig. 2A, lanes 1, 2, and 4). The presence of the histidine tag in v-opshis6x was confirmed by the following observations: 1) only v-opshis6x is recognized by the histidine tag antibody (see below), 2) the apparent molecular mass of v-opshis6x is larger (by about 2 kDa) than that of native opsin and v-ops (Fig. 2A, lane 3 versus lanes 2 and 4), 3) v-opshis6x does not react with the monoclonal antibody 1D4 (Fig. 2B, lane 1 versus 2), since the C-terminal histidine tag extension eliminates the epitope for this antibody (29Molday R.S. Progr. Retinal Res. 1989; 8: 173-209Crossref Scopus (68) Google Scholar). Hence, recombinant virus AcNPV/opshis directs expression of a fully intact histidine-tagged v-ops. The expression level varies between 20 and 30 pmol/106 cells, which is comparable to wild-type v-ops (6DeCaluwé G.L.J. VanOostrum J. Janssen J.J.M. DeGrip W.J. Methods Neurosci. 1993; 15: 307-321Crossref Scopus (22) Google Scholar). Regeneration and Purification of v-Rhohis6x—To convert expressed v-opshis6x into v-Rhohis6x, we incubated total membranes, harvested at 3 days post-infection from infected Sf9 cells, with 11-cis-retinal. Spectral analysis of regenerated samples showed that this resulted in 18 ± 2 pmol of v-Rhohis6x per 106 cells (n = 7). For purification by IMAC, regenerated samples were solubilized using DoM as a detergent and loaded onto a Ni2+-NTA agarose column. This column appeared to bind v-Rhohis6x with high capacity. Proteins, which have natural affinity for metal ions are bound as well. A variety of washing and elution conditions have been tested to remove this contamination as much as possible without sacrificing binding capacity of the column and recovery of v-Rhohis6x. Eluted fractions were analyzed by SDS-PAGE (protein pattern), immunoblotting (anti-rhodopsin CERNJS858 antiserum), and by UV/Vis spectroscopy (A500 to detect and quantify v-Rhohis6x; ratio A280/A500 as a purity indicator). Typical results are presented in Fig. 3 and Table I. Lane 1 in Fig. 3, A, B, and C, presents the total extract of Sf9 membranes after regeneration. Only by immunoblot analysis can glycosylated v-opshis6x be identified (arrow) together with a minor amount of unglycosylated species (arrowhead). The nonbound, flow-through fraction shows a very similar protein pattern (lane 2), except that most v-Rhohis6x has bound, since only minimal amounts of the glycosylated form are detected in this fraction. Additional washings with extraction buffer (buffer C) elute a complex protein population (Fig. 3, lanes 3 and 4), probably representing aspecifically or very weakly bound proteins. The nonglycosylated v-opshis6x is already strongly present in these fractions (Fig. 3, β and C, lanes 2–4, arrowhead) and apparently is not very well retained by the column. Protein contamination, weakly interacting with the column, could be eluted by raising the imidazole concentration to 25 mM (Fig. 3, lane 5). Under these conditions, a minor amount of v-Rhohis6x was eluted. Most of the specifically bound v-Rhohis6x, however, only was eluted upon raising the imidazole concentration to 100 mM (Fig. 3, lane 6). This fraction also contains a minor amount of nonglycosylated species and is still contaminated by several other minor bands (Fig. 3A, lane 6, open arrowheads).Table IGeneral characteristics of v-Rhohis6× purification by IMAC as determined by UV/Vis spectroscopyConditionBuffer CBuffer ESolubization (%)100 ± 20100 ± 8Nonbound (%)8 ± 55 ± 6Recovery (%)84 ± 480 ± 5A280/A5002.7 ± 0.42.4 ±0.4 Open table in a new tab The amount of nonglycosylated v-opshis6x in the DoM extract and column fractions varied between different experiments, but it was always present at immunodetectable levels (data not shown). In addition, immunoblots showed the presence of a third opsin species (Fig. 3B, lanes 1–4) which on SDS-PAGE gels migrated between the glycosylated and nonglycosylated v-opshis6x. This species did not react with the anti-histidine tag antibody (Fig. 3C, lanes 1–4). We were unable to detect this band in the final purified v-Rhohis6x fraction (Fig. 3B, lane 6). An overview of IMAC results obtained under two conditions, as determined by spectroscopic analysis, is given in Table I. Nearly complete binding of v-Rhohis6x to the Ni2+-NTA agarose was attained. At pH 8.0 (buffer C), a pH at which IMAC is usually performed, an average recovery of 84% was achieved with a purification factor of at least 450. An attempt to reduce contamination by weakly binding proteins, by applying the membrane extract in the presence of a higher imidazole concentration (10 mM; buffer D), resulted in a much higher loss of v-Rhohis6x and in fact lowered the purification factor (not shown). Finally, the performance of the procedure we developed at pH 8.0, was evaluated at pH 6.5, since rhodopsin and most of its mutants are thermally much more stable at the latter pH. This actually improved the purification factor to at least 500, without significant reduction in recovery (Table I, buffer E). At pH 6.5, best v-Rhohis6x samples had a purity of 80–85% (A280/A500 ratio of 2.0–2.1), and the average purity of combined fractions was 70–75%. The maximal capacity of the Ni2+-NTA agarose column for v-Rhohis6x has not been determined exactly but is at least 35 nmol/ml bed volume. The efficiency of this IMAC procedure is spectrally illustrated in Fig. 4A. Curve 1 represents the total membrane extract applied to the column, while curve 2 presents the spectrum of the combined purified v-Rhohis6x fractions. The contamination in the v-Rhohis6x fraction obtained after IMAC resides in several minor bands. These are largely removed upon subsequent reconstitution of v-Rhohis6x into retina lipid proteoliposomes, which represent a more native-like environment, we routinely use to analyze functional properties of recombinant rhodopsin (6DeCaluwé G.L.J. VanOostrum J. Janssen J.J.M. DeGrip W.J. Methods Neurosci. 1993; 15: 307-321Crossref Scopus (22) Google Scholar). To simplify reconstitution, the detergent used during regeneration (DoM) is exchanged during IMAC for nonylglucose, which is more easily exchanged for phospholipids (30DeGrip W.J. Olive J. Bovee-Geurts P.H.M. Biochim. Biophys. Acta. 1983; 734: 168-179Crossref Scopus (26) Google Scholar). The resulting v-Rhohis6x proteoliposomes contain at least 95% rhodopsin on a protein base and are suitable for all functional analyses (Fig. 3A, lane 8) Spectral Properties and Photocascade of v-Rhohis6x—Curve 2 in Fig. 4A is a typical absorbance spectrum of the combined v-Rhohis6x fractions obtained after IMAC purification. Illumination of v-Rhohis6x “bleaches” the main absorbance band at 500 nm, and we used difference spectra to calculate the λmax more accurately. Unexpectedly, the absorbance band of v-Rhohis6x turns out to be slightly red-shifted (λmax = 501 ± 1 nm, n = 7) relative to wild-type (λmax = 498 ± 1 nm) (Fig. 4S). This slight but significant red shift has been observed in all samples produced so far, both before and after IMAC purification. The shift is independent of the presence of 10 mm Ni2+ ions (complexed histidine tag) or 10 mm EDTA (free histidine tag). Analysis of the later part of the pho" @default.
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• W2103208001 title "Histidine Tagging Both Allows Convenient Single-step Purification of Bovine Rhodopsin and Exerts Ionic Strength-dependent Effects on Its Photochemistry" @default.
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