| Products/Services Used | Details | Operation |
|---|---|---|
| Codon Optimization | Codon optimized human AQP cDNAs for S. cerevisiae were purchased from Genscript, USA.The solubilized protein was diluted to a detergent concentration corresponding to 1.5 times CMC of the detergent used and incubated overnight in a beaker with Ni-resin (Genscript, USA) Non-solubilized material was pelleted at 70,000 rpm in the Beckmann Optima TL200 ultracentrifuge for 30 minutes at 4 °C. Solubilized membranes were diluted in buffer B with protease inhibitors to a detergent concentration corresponding to 1.5 times CMC and incubated overnight in a glass beaker with 1 ml of Ni-NTA Agarose (Genscript, Germany) at 4 °C with slow magnetic stirring. | Get A Quote |
The sparse number of high-resolution human membrane protein structures severely restricts our comprehension of molecular physiology and ability to exploit rational drug design. In the search for a standardized， cheap and easily handled human membrane protein production platform， we thoroughly investigated the capacity of S. cerevisiae to deliver high yields of prime quality human AQPs， focusing on poorly characterized members including some previously shown to be difficult to isolate. Exploiting GFP labeled forms we comprehensively optimized production and purification procedures resulting in satisfactory yields of all nine AQP targets. We applied the obtained knowledge to successfully upscale purificatio... More
