Expression of active human sialyltransferase ST6GalNAcI in Escherichia coli

Georgios Skretas,George Georgiou,Marc F Schwartz,Shawn Defrees,Sean Carroll,Karl F Johnson

doi:10.1186/1475-2859-8-50

Abstract

BackgroundThe presence of terminal, surface-exposed sialic acid moieties can greatly enhance the in vivo half-life of glycosylated biopharmaceuticals and improve their therapeutic efficacy. Complete and homogeneous sialylation of glycoproteins can be efficiently performed enzymically in vitro but this process requires large amounts of catalytically active sialyltransferases. Furthermore, standard microbial hosts used for large-scale production of recombinant enzymes can only produce small quantities of glycosyltransferases of animal origin, which lack catalytic activity.Results and conclusionIn this work, we have expressed the human sialyltransferase ST6GalNAc I (ST6), an enzyme that sialylates O-linked glycoproteins, in Escherichia coli cells. We observed that wild-type bacterial cells are able to produce only very small amounts of soluble ST6 enzyme. We have found, however, that engineered bacterial strains which possess certain types of oxidative cytoplasm or which co-express the molecular chaperones/co-chaperones trigger factor, DnaK/DnaJ, GroEL/GroES, and Skp, can produce greatly enhanced amounts of soluble ST6. Furthermore, we have developed a novel high-throughput assay for the detection of sialyltransferase activity and used it to demonstrate that the bacterially expressed ST6 enzyme is active and able to transfer sialic acid onto a desialylated O-glycoprotein, bovine submaxillary mucin. To the best of our knowledge, this is the first example of expression of active human sialyltransferase in bacteria. This system may be used as a starting point for the evolution of sialyltransferases with better expression characteristics or altered donor/acceptor specificities.

Highlights

The presence of terminal, surface-exposed sialic acid moieties can greatly enhance the in vivo half-life of glycosylated biopharmaceuticals and improve their therapeutic efficacy
Expression of ST6GalNAc I (ST6) in E. coli strains having an oxidizing cytoplasm ST6 catalyzes the transfer of N-acetylneuraminic acid (Neu5Ac - the most common type of sialic acid in higher animals) from the sugar donor CMP-Neu5Ac onto a terminal β-D-galactopyranosyl (Gal) residue of an O-linked glycoprotein to generate an α 2-6 linkage [8]
ST6 is a type II transmembrane glycoprotein, comprised of a short N-terminal cytosolic tail, a hydrophobic signal-anchor sequence that is embedded in the membrane, a so-called "stem" region, and a long C-terminal catalytic domain that is exposed to the lumen of the Golgi apparatus

Summary

Introduction

The presence of terminal, surface-exposed sialic acid moieties can greatly enhance the in vivo half-life of glycosylated biopharmaceuticals and improve their therapeutic efficacy. Most of the widely used bacterial expression hosts, such as Escherichia coli, cannot perform protein glycosylation [1] Eukaryotic hosts, such as yeast, insect, and non-human mammalian cell lines are capable of protein glycosylation, they introduce non-native glycosylation patterns that can result in undesired effects, including decreased biological potency and immunogenicity [1]. One of the most important examples of the influence of glycan structure on pharmacological properties of therapeutic proteins is the strong dependence of the serum half-life of a glycoprotein on the presence of sialic acid moieties attached to the terminal site of its glycans [2]. It has been shown that the in vivo half-life and biological (hematopoietic) activity of EPO is directly proportional to the number of sialic acid moieties attached to its four naturally occurring glycosylation sites [4]. Site-specific incorporation of additional glycosylation sites into the protein's sequence and the resulting increase in the number of attached sialic acids per molecule has been shown to increase the half-life of EPO [5]

Methods

Results

Discussion

Conclusion