BIOT 139 |
| A novel strategy to produce large 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 GPCR inclusion bodies was 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 (NK1) that was found to express at high levels in inclusion bodies. Given the ability to make high levels of inclusion bodies, finding the right solubilizing agent proved to be difficult because common agents such as 8 M urea or 6 M Guanidium HCl 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 inactive human neurokinin 1 (hNK1), biophysical characterization was performed in different surfactants in order to develop appropriate refolding strategies.
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Biophysical and Biomolecular Symposium: Challenges to Membrane Protein Production and Characterization
8:00 AM-10:55 AM, Tuesday, 12 September 2006 Hilton San Francisco -- Imperial B Ballroom, Oral
Division of Biochemical Technology |