Table 1.

Cell-free glycoprotein synthesis titers with OST-dependent glycosylation systems

Extract	Protein	~ Yield*	Quantitation method	Glycosylation machineryϮ	Notes	References
Tobacco	Luc	270	Luminescence	E	Not glycosylated	[13]
	GOx	7 U/mL	Colorimetric GOx activity assay	E	Multi-subunit, DSB required for activity	[13]
Insect	Luc	45	Luminescence	N/A	Not glycosylated	Promega TNT T7 kit
	gp120	25	WB densitometry	E		[137]
	EPO	5	Radioactive counting, autoradiography	E		[152]
CHO	Luc	50	Luminescence	E	Not glycosylated	[11]
	IgG	1, 9 (SC)	Radioactive counting, autoradiography	E	Multi-subunit, DSB required for activity	[134]
	EPO	5	Radioactive counting, autoradiography	E		[141]
	EPO	120 (SC)	ELISA	Supplemented via CHO-derived ER/Golgi microsomes		[44]
E. coli	sfGFP	600	Fluorescence	CjLLO, CjOST	Not glycosylated	[48]
	sfGFP-glyco	120	Fluorescence, WB densitometry	CjLLO, CjOST		[48]
	EPO	10	ELISA	CjLLO, CjOST	Non-native sequons	[57]
	MBP	20	Radioactive counting, WB densitometry	FtLLO, CjOST		[133]

^*All units are in μg/mL unless indicated. U/mL refers to active enzyme units per mL CFGpS reaction. All reactions were conducted in batch, unless denoted with ‘SC’ for semicontinuous reaction conditions

^ϮE denotes when extracts were enriched with the endogenous glycosylation machinery from the host strain. For E. coli systems, the OST and LLO recombinantly expressed in the strain prior to lysis are indicated

Abbreviations are: firefly luciferase (Luc), glucose oxidase (GOx), epidermal growth factor receptor (EGFR), HIV-1 envelope glycoprotein (gp120), superfolder green fluorescent protein (sfGFP) maltose binding protein (MBP), erythropoietin (EPO), Francisella tularensis (Ft), Campylobacter jejuni (Cj), disulfide bond (DSB), Western blot (WB), and enzyme-linked immunosorbent assay (ELISA)