Expression einer funktionellen rekombinanten Glykosyltransferase in Escherichia coli

Abstract

Recombinant protein therapeutics have revolutionised the treatment of many diseases and are produced using well-established expression systems based on bacteria, yeast, insect and mammalian cells. Approximately 40 % of therapeutic proteins are glycoproteins and therefore the post-translational attachment of sugar residues is required. However, the use of bacteria for expression of eukaryotic proteins is limited by the lack of post-translational modification pathways, including those for glycosylation. The development of an Escherichia coli-based model system for production of human glycoproteins could potentially lead to increased yields, as well as significant decreases in processing time and costs. Aim of this work was the expression of functional human-derived glycosyltransferase UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 2 (GalNAcT2) in a recombinant E. coli strain. The gene encoding amino acids 52-571 of GalNAcT2 was codon-optimised and subsequently inserted into a pET-23 derived expression vector encoding a polyhistidine-tag which was translationally fused to the N-terminus of the glycosyltransferase (HisDapGalNAcT2). The glycosyltransferase HisDapGalNAcT2 was produced in SHuffle® T7 host cells using a recently published expression system including the pre-expression of the sulfhydryl oxidase Erv1p and protein disulfide isomerase PDI. Soluble HisDapGalNAcT2 was purified using Ni-NTA-chromatography and subsequently analysed by SEC-MALS and CD spectroscopy. The enzymatic activity of purified HisDapGalNAcT2 was monitored using a colorimetric assay by using a model acceptor peptide or, alternatively, the granulocyte-colony stimulating growth factor (G-CSF). The successful in vitro glycosylations were assessed by MALDI-TOF-MS analysis and ESI-MS analysis. The results indicated the presence of N-acetylgalactosamine residues at several putative glycosylation sites of the model acceptor peptide and at one position of the acceptor peptide G-CSF.