Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae

Shiroishi, Mitsunori; Tsujimoto, Hirokazu; Makyio, Hisayoshi; Asada, Hidetsugu; Yurugi-Kobayashi, Takami; Shimamura, Tatsuro; Murata, Takeshi; Nomura, Norimichi; Haga, Tatsuya; Iwata, So; Kobayashi, Takuya

doi:10.1186/1475-2859-11-78

Microbial Cell Factories

Table 1 Summary of the expression level and functional activity of the 25 GPCRs expressed S. cerevisiae under different culture conditions in small-scale cultures

From: Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae

			α-factor (−)				α-factor (+)
		Temp	30 °C		20 °C		30 °C		20 °C
		DMSO	−	+	−	+	−	+	−	+
Acetylcholine receptor	hCHRM2	TE (mg/L)	1.5	1.5	1.7	1.6	0.53	0.78	0.62	0.56
		FE (pmol/mg)	0.03	0.12	0.05	0.21	0.08	0.28	0.30	0.66
Adenosine receptor	hADORA2A	TE (mg/L)	0.20	0.17	0.06	0.06	0.70	0.68	0.84	0.92
		FE (pmol/mg)	5.3	6.8	2.4	2.4	24	25	16	20
Adrenergic receptor	hADRB2	TE (mg/L)	1.5	1.6	0.76	0.93	0.81	1.0	0.91	0.71
		FE (pmol/mg)	0.06	0.07	0.10	0.18	0.36	0.40	0.52	1.1
Dopamine receptor	hDRD1	TE (mg/L)	0.53	0.81	0.75	0.9	0.44	0.62	0.54	0.57
		FE (pmol/mg)	0.07	0.02	0.05	0.01	0.01	0.03	0.01	0.04
	hDRD2	TE (mg/L)	0.21	0.12	0.26	0.24	0.21	0.32	0.34	0.31
		FE (pmol/mg)	0.37	0.58	0.72	1.57	0.35	1.09	1.6	3.2
	hDRD4	TE (mg/L)	2.0	2.5	1.1	1.3	0.78	0.79	0.8	1.3
		FE (pmol/mg)	0.21	0.25	0.19	0.3	0.22	0.34	0.17	0.17
Histamine receptor	hHRH1	TE (mg/L)	0.95	1.1	1.6	1.7	0.37	0.53	0.65	0.63
		FE (pmol/mg)	0	0	0.34	0.61	0	0.15	0	0
	hHRH3	TE (mg/L)	0.96	1.0	0.61	0.55	0.48	0.64	0.61	0.81
		FE (pmol/mg)	0.4	0.62	0.24	0.11	0.74	0.87	0.27	0.32
	hHRH4	TE (mg/L)	0.38	0.51	0.67	0.75	0.33	0.55	0.42	0.52
		FE (pmol/mg)	0.45	0.6	0.16	0.27	0.68	1.1	0.22	0.15
Neuropeptide Y	hNPY1R	TE (mg/L)	0.11	0.11	0.11	0.11	0.36	0.4	0.25	0.26
		FE (pmol/mg)	0	0	0	0	0	0.12	0.07	0.21
	hNPYR2	TE (mg/L)	0.51	0.54	0.24	0.23	0.18	0.22	0.2	0.19
		FE (pmol/mg)	0	0.14	0.05	0	0	0	0.03	0
	hNPYR4	TE (mg/L)	1.0	1.5	0.91	1.2	0.27	0.61	0.24	0.27
		FE (pmol/mg)	0.13	0.18	0.1	0.07	0.21	0.35	0	0
	hNPYR5	TE (mg/L)	0.31	0.46	0.82	0.89	0.34	0.51	0.52	0.61
		FE (pmol/mg)	0.04	0.02	0.07	0.13	0	0.01	0	0
Neurotensin receptor	hNTSR1	TE (mg/L)	0.59	1.0	0.87	0.93	0.52	0.8	0.83	1.1
		FE (pmol/mg)	0.15	0.15	0.15	0.15	0.32	0.43	0.3	0.78
	hNTSR2	TE (mg/L)	0.73	1.3	1.4	1.5	0.65	1.0	0.8	0.89
		FE (pmol/mg)	0.18	0.21	0.26	0.18	0.19	0.19	0.11	0.16
Opioid receptor	hOPRK1	TE (mg/L)	0.54	0.76	0.4	0.34	0.24	0.32	0.27	0.29
		FE (pmol/mg)	0	0.08	0.09	0.16	0	0.11	0.1	0.15
Prostanoid receptor	hPTGER2	TE (mg/L)	0.24	0.16	0.2	0.18	0.28	0.43	0.49	0.48
		FE (pmol/mg)	0	0	0.19	0	0	0.18	0.1	0.47
	hPTGER4	TE (mg/L)	0.11	0.15	0.09	0.11	1.4	1.7	1.5	1.4
		FE (pmol/mg)	0	0	0	0	0	0	0.4	0
	hTBXA2R	TE (mg/L)	0.52	0.63	0.26	0.26	0.21	0.21	0.34	0.32
		FE (pmol/mg)	0.15	0.23	0.05	0.02	0.03	0	0.78	0.7
Serotonin receptor	hHTR1B	TE (mg/L)	0.9	1.1	0.46	0.58	0.34	0.23	0.46	0.46
		FE (pmol/mg)	0.54	0.62	0.71	0.48	0.89	1.1	2.0	1.9
	hHTR1D	TE (mg/L)	0.34	0.53	0.67	0.76	0.29	0.3	0.33	0.22
		FE (pmol/mg)	0.06	0.03	0.33	0.46	0.46	0.52	1.7	1.6
	hHTR5A	TE (mg/L)	0.3	0.62	0.48	0.59	0.2	0.25	0.23	0.23
		FE (pmol/mg)	0.02	0.18	0	0.07	0.24	0.34	0	0
Tachykinin receptor	hTACR1	TE (mg/L)	1.1	1.7	1.1	1.1	0.67	0.83	0.65	0.57
		FE (pmol/mg)	0	0.45	0.14	0.02	0.60	0.59	0.09	0.15
	hTACR2	TE (mg/L)	1.4	1.7	2.5	2.2	0.46	0.71	0.96	0.86
		FE (pmol/mg)	0.40	0.85	1.1	2.9	0.75	0.65	1.3	3.8
	rTACR2	TE (mg/L)	0.11	0.10	0.06	0.04	0.56	0.86	1.0	1.1
		FE (pmol/mg)	0.25	0.2	0.08	0.06	1.3	1.9	5.2	7.4

“TE” represents the total expression level estimated from whole-cell GFP fluorescence (mg/L culture). The intensity of 200 μL of 1 μM purified yEGFP in the 96-well plate was 7500 [rfu]. Whole-cell fluorescence was measured in a 200-μL cell suspension by using cultures at the same cell density as the culture. Therefore, the expression level was calculated from: TE [mg/L] = (GFP counts of whole cell [rfu])/(7500*10⁶ [rfu*L/mol])* (molecular weight*10³ [mg/mol]). “FE” represents the functional activity from a single-point radioligand binding assay (pmol/mg membrane protein). Conditions highlighted in bold type are the best and second-best conditions for the GFP-based expression level and the functional activity.

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