Green fluorescent protein as a reporter of human μ-opioid receptor overexpression and localization in the methylotrophic yeast Pichia pastoris
Introduction
The human μ-opioid receptor (HuMOR) belongs to the super family of seven transmembrane segments G-protein coupled receptors (GPCRs). Responsible for specific interactions with endogenous opioid peptides and exogenous alkaloids such as morphine, this protein is involved in several fundamental biological processes such as pain perception, stress and emotions. A huge number of pharmaceutical substances mediate their effects through binding to GPCRs and thus, these proteins represent major targets for pharmaceutical applications (Stadel et al., 1997, Bikker et al., 1998). This pharmacological relevance of GPCRs has led to an increasing interest in their three-dimensional structure.
For all the seven transmembrane α-helices proteins, whether or not they belong to GPCRs super family, structural information is very sparse. At this time, high-resolution three-dimensional structures of only two of them are known: the ones of the light sensitive opsins found in high quantities in certain bacteria and in the retinae of higher organisms (Luecke et al., 1999, Palczewski et al., 2000). This lack of structural data mainly originates in the membranous nature of those proteins, and on their natural very low level of expression. For this reason, it is essential to develop more efficient heterologous expression systems.
Several organisms have been tested for the expression of the HuMOR, with varying results concerning the expression levels obtained (Stanasila et al., 1998). Among them, the methylotrophic yeast Pichia pastoris has been described as a particularly efficient host for high-level expression of heterologous proteins when their encoding genes are placed under control of the host alcohol oxidase-1 (AOX1) promoter (Cregg et al., 1993). The AOX1 promoter is highly inducible by methanol and tightly regulated. It is repressed in the presence of glucose or glycerol, and allows the production of 100 g of dry cell weight per litre of culture. In the presence of methanol, the promoter activity is maximal and AOX1 may contribute to 30% of the total protein content (Cregg et al., 1993). Since P. pastoris is eucaryotic, its intracellular environment is more suitable for expression and correct folding of eucaryotic proteins. This yeast was initially chosen in our laboratory to express the HuMOR (Talmont et al., 1996). This work was one of the first successes of a GPCR expression in P. pastoris. The protein was functional according to its ligands binding properties, expressed at a level of 0.4 pmol of receptor per mg of membrane proteins and could be 85%-perdeuterated (Massou et al., 1999). Other GPCRs have also been expressed in that yeast at higher expression levels, such as the β2-adrenergic and the 5HT5A-serotoninergic receptors (Weiss et al., 1995, Weiss et al., 1998a, Weiss et al., 1998b), thus definitely making P. pastoris an efficient host for the production of GPCRs. Nevertheless, considering the high efficiency of the AOX1 promoter, an expression level of 0.4 pmol of receptor per mg of membrane proteins-determined through radiolabelled-ligand saturation experiments using tritiated diprenorphine ([3H]-DPN), a μ-selective antagonist is not really conclusive. Among the different factors that are known to influence the ability of a given receptor to interact with its ligands, the non-appropriate lipidic environment of the receptor is particularly relevant (Nunez and Glass, 1982, Kirilovsky et al., 1987, Gimpl et al., 1995). It has already been reported that [3H]-DPN binding of the HuMOR was significantly affected by its surrounding lipidic environment (Hasegawa et al., 1987). In yeast, the major sterol of the plasma membrane is ergosterol. A recent study also seems to suggest that cholesterol and ergosterol could have quite opposite effects with respect to the ligand binding properties of the HuMOR (ergosterol was found to constrain the μ-opioid receptor in an inactive state, and could not replace cholesterol in activating it) (Lagane et al., 2000). In view of these observations, one can easily doubt in ligand binding studies ability to detect the total amount of receptor expressed within the cells.
In order to precisely quantify the expression level of the HuMOR in P. pastoris independently of ligand binding studies, the receptor was fused to an enhanced green fluorescent protein (EGFP). Fusion proteins have long been used to evaluate the expression of a particular gene of interest by fusing it to another reporter gene. The reporter must be a very stable protein that can be easily quantified and, because the two genes are fused, the expression of the reporter gene should accurately reflect the expression of the protein of interest. The GFP from jellyfish was chosen because of the numerous advantages it has over other reporter systems. This protein retains its ability to fluoresce in vivo when expressed in various organisms ranging from bacteria to Drosophila. Much more interesting is the stability of GFP fluorescence in the presence of strong denaturing agents such as SDS (for a recent review on GFP see Tsien, 1998). This last property of GFP is particularly relevant since many of the GPCRs lose their functionality in the presence of detergents during solubilization processes, so that a fusion to GFP allows following those molecules during this step. Several GPCRs have already been fused to GFP in their C-terminal part, but most studies have used the fusion proteins to study mainly receptors trafficking and agonist-mediated desensitisation and internalisation (for example, see Tarasova et al., 1997, Barak et al., 1997).
In the present work, GFP was fused to the N-terminal part of the HuMOR, and mainly used to quantify HuMOR effective expression levels. Our results clearly demonstrate that such an N-terminal tagging of the HuMOR with GFP does not modify the functional receptor expression levels and ligand binding properties, and that the GFP moiety could serve both as an in vivo reporter of membrane protein spatial localization, and as an in vitro reporter of HuMOR expression levels. In fact, we found that huge amounts of receptor are expressed within the cells that could never have been detected without the GFP strategy. Finally, we show here that GFP fluorescence property can be efficiently used as a generic tool to simplify many steps ranging from clones selection to product optimisation and monitoring.
Section snippets
Bacterial, yeast strains and media
Escherichia coli strain Top10F′ [(RecA) F {lacIqTn10(TetR)} mcrA Δ(mrr-hsdRMS-mcrBC)Φ80lacZΔM15ΔlacX74recA1 deoR araD139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG] was used for the propagation of recombinant plasmids. E. coli transformants were selected on low salt LB plates pH 7.5 (0.5% (w/v) yeast extract, 1% (w/v) tryptone, 0.5% (w/v) NaCl, 1.5% bacteriological agar) with 25 μg ml−1 Zeocin. P. pastoris wild type and strains GS115 (his4), SMD1163 (his4, pep4, prB1) (generous gift from
Expression of the HuMOR–c-myc–his-tag and GFP–HuMOR–c-myc–his-tag proteins in P. pastoris
After electrotransformation of the four different P. pastoris strains with the αMF–HuMOR–c-myc–his-tag and the αMF–GFP–HuMOR–c-myc–his-tag fusion genes, Zeocin resistant transformants were selected, and tested for the expression of the fusion proteins. This was done using the binding of the opioid antagonist [3H]-DPN for the HuMOR–c-myc–his-tag construction, while it was performed using GFP fluorescence for the GFP–HuMOR–c-myc–his-tag chimeric receptor as described below. For the two coding
Discussion
Three different sequences were fused to the N- and/or to the C-terminal end of the HuMOR receptor: the GFP gene was added to the N-terminal part of the HuMOR in order to use fluorescence measurements during the recombinant clones selection step and to quantify chimeric receptor expression levels; Moreover, in order to detect the expression of the protein by Western blotting and to further purify the μ-opioid receptor, we have both fused a c-myc epitope and a six histidines tag (his-tag) to the
Acknowledgements
This work was supported by a grant (to V. Sarramegna) from the Ministére de l'Education Nationale, de la Recherche et de la Technologie, by the Centre National de la Recherche Scientifique, by the Université Paul Sabatier (Toulouse III), and by the Conseil Régional de la Région Midi-Pyrénées. We wish to thank Marie-Ange Dupont for all the help provided in electron microscopy experiments, and Jacques Feliu for DNA sequencing. Special thanks are also expressed to Dr L. Emorine and Pr. Jean Cros
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