G-protein coupled receptors (GPCRs) are a class of seven transmembrane helical proteins that are important drug targets for various diseases such as cancer, diabetes, HIV infection and depression. Despite their vast importance there is little detailed high-resolution structural information known about them. Currently, there is only one x-ray crystal structure of a GPCR, bovine rhodopsin, which was possible due to its natural abundance. The lack of high-resolution structural information hinders rational drug discovery efforts. A major bottleneck in obtaining structural information is heterologous expression capable of producing milligram quantities of GPCRs.
A novel strategy to produce milligram quantities of GPCRs is to express them as inclusion bodies in E. coli, and refold them into active, functional GPCRs. This strategy has some advantages over current methods for heterologous expression in yeast and mammalian hosts. These include the availability of strains, low cost of production, ease of use, and potential for scale up. Also, producing protein as inclusion bodies in E. coli reduces the toxicity of expression, with the potential production of 10’s of mgs of protein per liter of culture. Surprisingly, making GPCRs inclusion bodies was determined to be a non-trivial task. After screening different conditions such as growth temperature, cell lines, and various types of GPCRs, we identified one GPCR, neurokinin 1 (NK1R) that was found to express at high levels in inclusion bodies.
Given the ability to make high levels of inclusion bodies of hNK1R, finding the right solubilizing agent proved to be difficult because common agents such as 8 M urea or 6 M guanidium Cl failed to solubilize the receptor. A surfactant had to be identified that not only could solubilize the inclusion body, but would also be compatible with metal affinity chromatography. Upon screening, fos-choline 16 was identified to fit both criteria. With the ability to purify > 5 mg/L of culture (hNK1R), biophysical characterization was performed in different surfactants in order to develop appropriate refolding strategies. The inactive state of the hNK1R had significant secondary structure, as determined by circular dichroism, although it appeared to have no long-range interactions.
In order to determine why many GPCRs cannot be readily expressed as inclusion bodies, a time course expression of the human adenosine A2a receptor (hA 2aR) was performed in E. coli. We determined that the protein was being expressed as insoluble protein, then degraded. Further investigation shows that the expression of GPCRs as inclusion bodies activated the σ 32 (heat shock) response. This response upregulates chaperones and proteases which assist in protein folding and degradation. We then determined through expression of hA2aR in protease knockout strains that the heat shock protease ClpP was responsible for the degradation and thus the lack of expression.