We've updated our Privacy Policy to make it clearer how we use your personal data.

We use cookies to provide you with a better experience. You can read our Cookie Policy here.

Advertisement
Platform for the Rapid Construction and Evaluation of GPCRs for Crystallography in Saccharomyces Cerevisiae
News

Platform for the Rapid Construction and Evaluation of GPCRs for Crystallography in Saccharomyces Cerevisiae

Platform for the Rapid Construction and Evaluation of GPCRs for Crystallography in Saccharomyces Cerevisiae
News

Platform for the Rapid Construction and Evaluation of GPCRs for Crystallography in Saccharomyces Cerevisiae

Read time:
 

Want a FREE PDF version of This News Story?

Complete the form below and we will email you a PDF version of "Platform for the Rapid Construction and Evaluation of GPCRs for Crystallography in Saccharomyces Cerevisiae"

First Name*
Last Name*
Email Address*
Country*
Company Type*
Job Function*
Would you like to receive further email communication from Technology Networks?

Technology Networks Ltd. needs the contact information you provide to us to contact you about our products and services. You may unsubscribe from these communications at any time. For information on how to unsubscribe, as well as our privacy practices and commitment to protecting your privacy, check out our Privacy Policy

Background:
Recent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification. Therefore, the quick screening of functionally expressed stable receptor variants is vital.

Results:
We developed a platform using Saccharomyces cerevisiae for the rapid construction and evaluation of functional GPCR variants for structural studies. This platform enables us to perform a screening cycle from construction to evaluation of variants within 6-7 days. We firstly confirmed the functional expression of 25 full-length class A GPCRs in this platform. Then, in order to improve the expression level and stability, we generated and evaluated the variants of the four GPCRs (hADRB2, hCHRM2, hHRH1 and hNTSR1). These stabilized receptor variants improved both functional activity and monodispersity. Finally, the expression level of the stabilized hHRH1 in Pichia pastoris was improved up to 65 pmol/mg from negligible expression of the functional full-length receptor in S. cerevisiae at first screening. The stabilized hHRH1 was able to be purified for use in crystallization trials.

Conclusions:
We demonstrated that the S. cerevisiae system should serve as an easy-to-handle and rapid platform for the construction and evaluation of GPCR variants. This platform can be a powerful prescreening method to identify a suitable GPCR variant for crystallography.

The article is published online in the journal Microbial Cell Factories and is free to access.

Advertisement