Biochemical characterization of a glycosyltransferase Gtf3 from Mycobacterium smegmatis : a case study of improved prote
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ORIGINAL ARTICLE
Biochemical characterization of a glycosyltransferase Gtf3 from Mycobacterium smegmatis: a case study of improved protein solubilization Mahfoud Bakli1,2,4 · Loukmane Karim3 · Nassima Mokhtari‑Soulimane2 · Hafida Merzouk2 · Florence Vincent4 Received: 4 June 2020 / Accepted: 8 September 2020 / Published online: 16 September 2020 © King Abdulaziz City for Science and Technology 2020
Abstract Glycosyltransferases (GTs) are widely present in several organisms. These enzymes specifically transfer sugar moieties to a range of substrates. The processes of bacterial glycosylation of the cell wall and their relations with host–pathogen interactions have been studied extensively, yet the majority of mycobacterial GTs involved in the cell wall synthesis remain poorly characterized. Glycopeptidolipids (GPLs) are major class of glycolipids present on the cell wall of various mycobacterial species. They play an important role in drug resistance and host–pathogen interaction virulence. Gtf3 enzyme performs a key step in the biosynthesis of triglycosylated GPLs. Here, we describe a general procedure to achieve expression, purification, and crystallization of recombinant protein Gtf3 from Mycobacterium smegmatis using an E. coli expression system. We reported also a combined bioinformatics and biochemical methods to predict aggregation propensity and improve protein solubilization of recombinant Gtf3. NVoy, a carbohydrate-based polymer reagent, was added to prevent protein aggregation by binding to hydrophobic protein surfaces of Gtf3. Using intrinsic tryptophan fluorescence quenching experiments, we also demonstrated that Gtf3-NVoy enzyme interacted with TDP and UDP nucleotide ligands. This case report proposes useful tools for the study of other glycosyltransferases which are rather difficult to characterize and crystallize. Keywords Glycosyltransferase · Expression and purification of recombinant protein · Protein solubilization · Mycobacterium smegmatis
Introduction During the last 4 decades, the number of recombinant proteins used for several academic, medical, and industrial applications has increased dramatically (Warne and Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13205-020-02431-x) contains supplementary material, which is available to authorized users. * Mahfoud Bakli [email protected] Loukmane Karim [email protected] Nassima Mokhtari‑Soulimane [email protected] Hafida Merzouk [email protected] Florence Vincent [email protected]‑mrs.fr
Mahler 2018). This engineering field has been growing essentially due to considerable progress in available sequenced genomes, and to biotechnology and strategy developments in achieving high-level protein expression. It ranges from expression vector design to final application (Vandermies and Fickers 2019; Kushwaha and Salis 2015; Rosano and Ceccarelli 2014), during which several obstacles may be encountered. Some problems are related to intrinsic physicochemical features s
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