High level in vivo mucin-type glycosylation in Escherichia coli

Abstract

BackgroundIncreasing efforts have been made to assess the potential of Escherichia coli strains for the production of complex recombinant proteins. Since a considerable part of therapeutic proteins are glycoproteins, the lack of the post-translational attachment of sugar moieties in standard E. coli expression strains represents a major caveat, thus limiting the use of E. coli based cell factories. The establishment of an E. coli expression system capable of protein glycosylation could potentially facilitate the production of therapeutics with a putative concomitant reduction of production costs.ResultsThe previously established E. coli strain expressing the soluble form of the functional human-derived glycosyltransferase polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2) was further modified by co-expressing the UDP-GlcNAc 4-epimerase WbgU derived from Plesiomonas shigelloides. This enables the conversion of uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) to the sugar donor uridine 5′-diphospho-N-acetylgalactosamine (UDP-GalNAc) in the bacterial cytoplasm. Initially, the codon-optimised gene wbgU was inserted into a pET-derived vector and a Tobacco Etch Virus (TEV) protease cleavable polyhistidine-tag was translationally fused to the C- terminus of the amino acid sequence. The 4-epimerase was subsequently expressed and purified. Following the removal of the polyhistidine-tag, WbgU was analysed by circular dichroism spectroscopy to determine folding state and thermal transitions of the protein. The in vitro activity of WbgU was validated by employing a modified glycosyltransferase assay. The conversion of UDP-GlcNAc to UDP-GalNAc was shown by capillary electrophoresis analysis. Using a previously established chaperone pre-/co- expression platform, the in vivo activity of both glycosyltransferase GalNAc-T2 and 4-epimerase WbgU was assessed in E. coli, in combination with a mucin 10-derived target protein. Monitoring glycosylation by liquid chromatography electrospray ionization mass spectrometry (LC–ESI–MS), the results clearly indicated the in vivo glycosylation of the mucin-derived acceptor peptide.ConclusionIn the present work, the previously established E. coli- based expression system was further optimized and the potential for in vivo O-glycosylation was shown by demonstrating the transfer of sugar moieties to a mucin-derived acceptor protein. The results offer the possibility to assess the practical use of the described expression platform for in vivo glycosylations of important biopharmaceutical compounds in E. coli.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1013-9) contains supplementary material, which is available to authorized users.

New tools for recombinant protein production in Escherichia coli: A 5‐year update

“…Also, tools allowing for post‐translational modifications are still scarce. Even though glycosylation patterns can be obtained using engineered E. coli strains, more research is warranted in this area. Finally, even though the popularity of E. coli as a host is high, other microorganisms should be considered as options, such as Bacilli strains, Pseudomonas fluorescens , Corynebacterium glutamicum , and many others.…”

Development of dual‐inducible duet‐expression vectors for tunable gene expression control and CRISPR interference‐based gene repression in Pseudomonas putida KT2440

“…An appropriate promoter–repressor system plays a crucial role in designing metabolic engineering strategies in situations where the product of some genes is required in a lower amount, and a higher product is desired for the other genes (Cook et al ., 2018 ). In such cases, a more potent promoter is used to express the gene(s) whose product is required in a high amount or vice versa (Mueller et al ., 2018 ). The quantification of fluorescence signals revealed the promoter's strength for gene expression in the following order: P bad > P tac > P tetR/tetA .…”

“…To overcome the limitations presented by one inducible promoter system, researchers normally use two inducible systems simultaneously, in the form of either one plasmid (dual‐inducible approach) or two plasmids, each plasmid harbouring a different inducible promoter system (Gauttam et al ., 2019a ). Such an approach is useful in situations where one gene product is required to stimulate the expression of another gene, as in the case of co‐chaperone expression (Mueller et al ., 2018 ). For this purpose, duet‐expression vectors were constructed in Pseudomonas fluorescens (Nakata, 2017 ), P. putida (Yu et al ., 2018 ) and Corynebacterium glutamicum (Gauttam et al ., 2019b ).…”

Applications of catalyzed cytoplasmic disulfide bond formation

“…Several Golgi located glycosyltransferases have been expressed in the cytoplasm of E.coli using active mechanisms of disulfide bond formation to facilitate functional expression. These include B4GalT1 [27], ST3Gal1 [28], ST6Gal1 [28] and, of relevance to mucin-like O-glycosylation, GalNacT2 [25,29,30].…”