Mammalian Expression

The optimal hosts for mammalian GPCR expression are obviously mammalian cells due to the capabilities of their more native post-translational modification mechanisms and the presence of mammalian G proteins for functional activity assessments. Transient expression has been achieved for many GPCRs, such as adrenergic, dopamine, muscarinic, serotonin, and somatostatin receptors with Bmax values in the range of 10 to 20 pmol/mg. Although the expression levels have been acceptable, the transfection procedure has been inefficient and adaptation to large-scale production has been especially difficult.50 Recent development of a modified calcium-phosphate co-precipitation method has generated yields of hundreds of milligrams of a monoclonal antibody in 100 L suspension cultures of HEK293 EBNA cells.51 Although this bodes well for future receptor expression, the yields of the topologically more demanding GPCRs may continue to be less encouraging.

BHK, CHO, and HEK293 are the most frequently used cell lines for generation of stable expression of GPCRs. Stable expression in mammalian cells has been hampered by time-consuming processes and also by relatively low expression levels. However, stable overexpression of the human p2 AR in CHO and HeLa cell lines from vectors containing the gene for dihydrofolate reductase allowed a stepwise increase of methotrexate concentration, which substantially enhanced expression levels.52 Another problem has been the concern of instability of stable constructs as there is, despite antibiotic selection, a pressure to remove integrated foreign gene sequences. To address this question, inducible expression vectors based on tetracy-cline regulation have been constructed.53

Another approach involved a cold-inducible expression system based on the Sindbis virus replicon that generates no expression at 37°C and high levels below 34°C.54 Inducible systems have allowed expression of various GPCRs in the range of 5 to 20 pmol receptor/mg protein.

Viral vectors have proven useful due to their broad host ranges and strong promoters that often generate extreme expression levels. Adenovirus vectors have been applied for GPCR expression in rabbit ventricular myocytes, where binding activity in the range of 30 to 40 pmol/mg was obtained for the human p2 AR.55 Poxviruses, especially the hybrid bacteriophage vaccinia virus vector with the T7 promoter, have been used widely.56 Vaccinia-based GPCR expression of the neuropeptide Y (NPY) receptor generated 5 to 10 million receptors/cell.57

Probably the most frequently used heterologous viral gene expression system for membrane proteins is based on the Semliki Forest virus (SFV), a member of the single-stranded RNA alphaviruses. SFV vectors have been applied for more than 100 GPCRs and the expression levels in most cases were extremely high when measured by metabolic labeling and Western blotting.58

The binding activity of GPCRs on both whole cells and isolated membranes has been impressive, with Bmax values up to 200 pmol/mg protein. The broad host range of SFV has allowed expression studies in parallel in different mammalian host cells. Interestingly, the human neurokinin-1 receptor (hNKIR) was best expressed in CHO-K1 cells,59 whereas the m opioid receptor showed the highest binding activity in the rat C6 glioma cell line.60

The optimal time of expression varied strongly from one receptor to another. A large number of mutants of hNKIR61 and human dopamine D3 receptors (hD3R)62 expressed from SFV vectors were analyzed for binding and functional activities in comparison to wild-type receptors. The results were verified to the molecular models for hNKIR and hD3R, respectively, based on the high resolution structure of bac-teriorhodopsin. Interestingly, although the binding results for many mutants were in agreement with the postulated model, others suggested that the models need to be refined. The SFV system has been applied for large-scale receptor production in suspension cultures in fermenters and spinner flasks.56

Production of hNKIR as a fusion protein with the autocatalytic SFV capsid protein in CHO suspension cultures resulted in 10 mg/L of recombinant receptor. Purification of the C-terminally His6-tagged hNKIR generated homogenous active receptor. Within the MePNet structural genomics program, 100 GPCRs were expressed from SFV with a success rate of 96% (see Chapter 14).

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