Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae.
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.
Iwata Human Receptor Crystallography project, ERATO, JST, Kyoto 606-8501, Japan. shiroish@phar.kyushu-u.ac.jp
Mitsunori
M
Shiroishi
Hirokazu
H
Tsujimoto
Hisayoshi
H
Makyio
Hidetsugu
H
Asada
Takami
T
Yurugi-Kobayashi
Tatsuro
T
Shimamura
Takeshi
T
Murata
Norimichi
N
Nomura
Tatsuya
T
Haga
So
S
Iwata
Takuya
T
Kobayashi
D003460:Crystallization; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010843:Pichia / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011969:Receptors, Histamine H1 / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D043562:Receptors, G-Protein-Coupled; D011969:Receptors, Histamine H1; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins