A prokaryote-based cell-free translation system that efficiently synthesizes glycoproteins

Abstract

Asparagine-linked (N-linked) protein glycosylation has been observed in all domains of life, including most recently in bacteria and is now widely considered a universal post-translational modification. However, cell-based production of homogeneous glycoproteins for laboratory and preparative purposes remains a significant challenge due in part to the complexity of this process in vivo. To address this issue, an easily available and highly controllable Escherichia coli-based cell-free system for the production of N-linked glycoproteins was developed. The method was created by coupling existing in vitro translation systems with an N-linked glycosylation pathway reconstituted from defined components. The translation/glycosylation system yielded efficiently glycosylated target proteins at a rate of hundreds of micrograms/milliliters in half a day. This is the first time a prokaryote-based cell-free protein synthesis system has generated N-linked glycoproteins.

Modification of Streptococcus mutans Cnm by PgfS Contributes to Adhesion, Endothelial Cell Invasion, and Virulence

“…It is worth mentioning that a recombinant form of Cnm expressed in E. coli migrated at the same mobility as Cnm expressed by the ⌬pgfS strain (ϳ90 kDa) (data not shown). It is highly unlikely that the laboratory strains of E. coli possess the necessary machinery to glycosylate Cnm (38). Thus, such a discrepancy between the electrophoretic mobility and the predicted mass is likely due to the unique biochemical properties of the B domain of Cnm, a highly acidic repeat region with a negative net charge (pI Ͻ 3.75).…”

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

“…67,68 Furthermore, glycosylated proteins could possibly be www.tandfonline.com 237 mAbs produced by including appropriate enzymes in cell-free reactions. 69 Alternatively, a non-natural amino acid can be incorporated at the glycosylation site N297, 49 followed by site-specific conjugation of glycans in a post-translation manner.…”

Structural Biology of Glycoproteins

“…Therefore, production of glycoproteins is most often performed using eukaryotic cells, although the aglycosylated protein can be expressed as inclusion bodies in E. coli and refolded and also produced using cell-free systems. Recent advances in glycosylation pathway engineering have resulted in both E. coli [7] and cell-free systems [8] which are capable of introducing Nglycosylation onto a protein. These methods are discussed in the relevant sections below.…”

“…These systems give glycosylation patterns native to the host which in the case of insect cells can be modified using endoglycosidases and in the case of mammalian cells can be blocked using inhibitors (Section 3.4.1). Recently both the E. coli lysate cell free synthesis system and the PURE system for in vitro translation using purified components of E. coli [66] have been adapted for production of glycoproteins [8]. In this article, Guarino and DeLisa used the protein glycosylation locus from Campylobater jejuni to supplement both systems and produce glycoprotein with the GlcGalNAc5Bac (where Bac represents bacillosamine) glycosylation pattern of C. jejuni ( Figure 5) [8].…”

“…Recently both the E. coli lysate cell free synthesis system and the PURE system for in vitro translation using purified components of E. coli [66] have been adapted for production of glycoproteins [8]. In this article, Guarino and DeLisa used the protein glycosylation locus from Campylobater jejuni to supplement both systems and produce glycoprotein with the GlcGalNAc5Bac (where Bac represents bacillosamine) glycosylation pattern of C. jejuni ( Figure 5) [8]. Cellfree systems have yet to yield a glycosylated protein for which a structure has been deposited in the PDB.…”