Eukaryotic Membrane Proteins

Membrane proteins are extremely difficult to overexpress in an heterologous system. While E.Coli serves as a perfect host for expression, various difficulties render this system as an inconvenient tool of use.

Bacterial expression based on Escherichia coli has been one of the most widely studied because of its ease of availability, handling and scale up; and furthermore it is cheap to grow. However, this system has turned out not to be suitable for expression of mammalian transmembrane proteins due to the lack of post-translational modifications (e.g. glycosylation, fatty acid acylation, and phosphorylation) and the processing events required for correct folding of mammalian proteins. The
accumulation of expressed proteins in inclusion bodies (in a misfolded state) is yet
another drawback, as it is very difficult to recover functional protein. In addition,
insertion of the recombinantly expressed mammalian proteins into bacterial membranes
has caused toxicity in host cells.

YEASTS
Yeasts are unicellular eukaryotes which can easily be grown in large-scale cultures. They combine the advantages of short generation times (2 h) and growth in simple media. Easy handling and rapid genetic manipulation and the potential to perform eukaryotic post-translational modification renders yeast as one of the organisms of choice for over-expression. High levels of heterologous expression in yeast have been obtained with β2-adrenergic receptor, with the expression level being 115 pmol/mg of the membrane protein, for an O.F. 23. However, the level of expression of other GPCRs expressed in this system varies between 0.02 pmol/mg and 40 pmol/mg. Though yeast has been rather widely used for production of membrane proteins and GPCRs, there are four important issues. First, the yeast is surrounded by a tough cell wall which must be disrupted in order to extract the membranes and
intracellular proteins. In addition, protease-deficient strains should be used and protease inhibitors should be included during breakage of the cells. Second, the lipid composition of yeast membranes is different from that of mammalian cells. The low cholesterol content or even the presence of ergosterol results in altered binding properties of the expressed membrane proteins. Third, though the yeast system is capable of performing eukaryotic post-translational modification, the composition and quantity of N-glycans added by yeast are different from that of mammalian cells, and this can be problematic when specific oligosaccharide structures are essential for functionality. (Fourth and) finally, during large scale production, an appropriate ligand should be added to the growth medium and the pH should be maintained at the optimal value for the receptor in study.

A few other systems have been used for the expression of membrane proteins and GPCRs. Archaebacteria such as Halobacterium salinarium have been used to express two receptors successfully, the β2-adrenergic receptor and the yeast α-mating factor receptor. H.salinarium has the capacity to express large quantities of bacteriorhodopsin; and it was assumed that this system could express other transmembrane mammalian proteins. However, this was not the case and even chimeras of bacteriorhodopsin and mammalian transmembrane proteins resulted only in modest expression levels.

In addition to Baculovirus/insect cell, there are other insect systems.
For instance, the extracellular part of the luteinizing hormone (LH receptor) has been expressed in a non-functional form in a caterpillar expression system. In addition, there is a new system for expression of GPCRs in fly eyes. The Drosophila melonogaster metabotropic glutamate receptor (DmGluRA) was expressed by this novel strategy in good yields (170 μg/g fly heads) and was purified to homogeneity.

Insect Cells / BACULOVIRUS

The insect cell based baculoviral system provides an alternative approach for expression of GPCRs. The advantage of the baculovirus expression system is that it expresses the majority of membrane proteins and GPCRs in a functional form and at levels 10-100 fold higher than those observed in cells and tissues endogenously expressing them. In this system, the protein production results from infection of insect cells by recombinant viruses encoding the gene(s) of interest. The Baculovirus Autographica californica multiple nuclear polyhedrosis virus (AcMPPV) has been most widely used for the expression of membrane proteins in insect cells (usually using Spodoptera frugiperda, Sf9 cells). In most cases, the expression is driven by the polyhedrin or the p10 promoter, both of which are ‘very late’ promoters and are switched on only about 24 h post viral infection. The expression of the recombinant proteins under the control of very late promoters generally peaks 48-72 h post infection. The virus undergoes a lytic cycle and the cells die about 4-5 days post infection. The insect cells are easy to grow, can adapt to serum-free media and growth can be scaled up with ease. These cells can also perform post-translational modifications such as fatty acid acylation, phosphorylation and glycosylation. The recombinant proteins exhibit characteristics very similar to their native counterparts. In addition, co-expression of GPCRs and mammalian G-proteins is possible in this system allowing in vivo analysis of the specificity of interactions with G protein subtypes. A large number of membrane proteins and GPCRs have been successfully expressed using this system with levels as high as 10-
100 pmol/mg of membrane protein. The receptors which were recombinantly expressed
at such high levels include β-adrenergic, serotonin, muscarinic, dopamine, neurokinin
and chemokine receptors.
The expression levels sometimes depend on receptor subtype as well as the presence and position of a fusion tag. One drawback of this system is that it
often gives rise to heterogeneous proteins, arising out of poor glycosylation, which results in altered binding properties. Also, due to low cholesterol abundance in insect cells, alteration in GPCR binding has been observed with oxytocin receptor. Coexpression of various chaperones, peptidases, foldases and glycosylating enzymes has been shown to improve the secretion, processing and glycosylation of heterologous
proteins expressed in the baculovirus system.