TABLE 81 Continued Expression Systems for GPCRs



Disadvantages Target GPCR Ref.

Eukaryotic —


Viral, Mammalian

Broad host range Slow virus production

Vaccinia virus SFV

High expression Safety concerns

Broad host range Relatively expensive

Extreme expression Safety concerns

>100 GPCRs

MB, membrane bound; IB, inclusion bodies; SFV, semliki forest virus.

a Unpublished results, M. Shirouzu, RIKEN Institute, Yokohama, Japan. b Unpublished results, R. Wagner, University of Strasbourg, France.

satisfactory levels in bacteria due to the toxic effects they cause to host cells and the difficulties in refolding when expressed in inclusion bodies. Despite these problems, some success has been achieved for expression of hA2aR and rNTR in bacterial inner membranes and refolding of LT1R from inclusion bodies.

Nevertheless, these achievements have required many years of hard work, and it seems that the methods developed are relatively receptor specific and not easily applicable to a large number of GPCRs. One shortcoming of bacterial expression of mammalian GPCRs is the lack of appropriate post-translational modification capacity. For instance, many GPCRs are glycosylated, a mechanism not present in E. coli. On the other hand, glycosylation may not be required for successful crystallization and may even interfere with appropriate crystal formation.

Expression of mammalian recombinant proteins in various yeast strains has been very successful due to their eukaryotic status with post-translational mechanisms similar to those present in mammalian cells. However, yeast cells are quite different from their mammalian counterparts in respect to glycosylation patterns and hyper-glycosylation is a typical phenomenon. GPCR expression has been achieved at relatively high levels in S. cerevisiae and S. pombe, but clearly P. pastoris generated the best expression levels. Several GPCRs have been expressed at high levels and clearly the very high biomasses obtained in fermenter cultures provide good bases for obtaining large quantities of receptors. The value of the purification procedure has to some extent been hampered by the difficulties in breaking the thick walls of yeast cells.

Insect cells have provided robust expression of GPCRs, especially by application of baculovirus vectors. The advantages include the presence of mammalian-like post-translational mechanisms, although certain differences exist between insect and mammalian cells. Because insect cell lines grow in semi-attached cultures, scale-up has been straightforward, albeit more expensive than for bacteria and yeasts. As previously described, record high binding activity could be obtained for the truncated non-glycosylated form of the turkey p-adrenergic receptor. Time will tell whether glycosylation requirements are essential in crystallography. The establishment of stable cell lines in Drososphila Schneider-2 cells looks also potentially promising.

Recombinant expression in mammalian cells is the closest approach to native mammalian GPCRs, but it has been hampered by low yields and time-consuming and expensive procedures. Improvements in transfection methods for transient expression and novel inducible vectors for stable expression have produced some progress. Alternatively, viral vectors, especially SFV vectors have provided both rapid high level expression in a broad range of host cells and an established method for large scale production. The drawbacks with SFV are the relatively high costs for virus production and the need to address safety concerns related to application of infectious, albeit replication-deficient, recombinant virus particles.

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