Cell-free extract preparation protocol for enrichment of membrane vesicles and increased glycoprotein yields

A method of synthesizing and glycosylating a target protein in a cell-free glycoprotein synthesis (CFGpS) reaction in vitro, the method comprising:

• contacting an expression vector encoding the target protein to an Escherichia coli (E. coli) cell S12 homogenized lysate, wherein the E. coli cells are engineered to express heterologous glycosylation machinery:
• i) transcribing and translating the target protein in the E. coli S12 homogenized lysate;
• and
• ii) glycosylating the target protein in the E. coli S12 homogenized lysate,
• wherein the lysate comprises membrane vesicles enriched in the heterologous glycosylation machinery as compared to an S30 E. coli homogenized lysate, or an S12 or S30 sonicated E. coli lysate.