PUREfrex® is a cell-free protein synthesis system based on the PURE system, which is reconstructed from only the factors involved in protein synthesis in E. coli. Therefore, it is easy to be applied for the post-translational modification of the synthesized protein controlling the reaction conditions and additional factors. So far, we have shown that it is possible to synthesize active disulfide bond-containing proteins by adding an oxidizing agent and protein disulfide isomerase (PDI). Here, we investigated whether it is possible to synthesize glycoproteins with N-linked sugar chain using PUREfrex® with glycosyltransferases and substrate sugar donors.

The glycosylation reaction was performed with reference to the method of Jewtett et al (Kightlinger et al. (2019) Nat. Commun., 10, 5404). A model protein (Im7-6) derived from E. coli Colicin-E7 immunity protein in which an N-linked glycosylation site is inserted, and several types of glycosyltransferases were synthesized with PUREfrex®, and these were mixed with sugar donors to perform an in vitro glycosylation (IVG) of Im7-6. As a result of SDS-PAGE and mass spectrometry after IVG, enzyme-dependent addition of galactose, GlcNAc or sialic acid was confirmed starting from the addition of glucose to the asparagine (Asn) residue in the glycosylation site of Im7-6. Furthermore, when a one-pot cell-free protein synthesis-glycosylation reaction (CFPS-IVG one-pot) was carried out in which Im7-6 and glycosyltransferase were synthesized and glycosylated at the same time, glycosylated Im7-6 was confirmed by this method as well. It was also found that glycosylation is possible for disulfide bond proteins and membrane proteins by optimizing the IVG method.

From the results above, it was shown that glycoprotein can be synthesized with PUREfrex® and that it can be a good system to evaluate post translational modification.