Biology subjects

Jewett, M. C.

Publications and source records attributed to Jewett, M. C..

57 records · Page 4Linked to original sources

bioRxiv2017

Diversity of translation initiation mechanisms across bacterial species is driven by environmental conditions and growth demands

The Shine-Dalgarno (SD) sequence is often found upstream of protein coding genes across the bacterial kingdom, where it enhances start codon recognition via hybridization to the anti-SD (aSD) sequence on the small ribosomal subunit. Despite widespread conservation of the aSD sequence, the proportion of SD-led genes within a genome varies widely across species, and the evolutionary pressures shaping this variation remain largely unknown. Here, we conduct a phylogenetically-informed analysis and show that species capable of rapid growth have a significantly higher proportion of SD-led genes in their genome, suggesting a role for SD sequences in meeting the protein production demands of rapidly growing species. Further, we show that utilization of the SD sequence mechanism co-varies with: i) genomic traits that are indicative of efficient translation, and ii) optimal growth temperatures. In contrast to prior surveys, our results demonstrate that variation in translation initiation mechanisms across genomes is largely predictable, and that SD sequence utilization is part of a larger suite of translation-associated traits whose diversity is driven by the differential growth strategies of individual species.

systems biology↗

bioRxiv2017

A pipeline for interrogating and engineering single-subunit oligosaccharyltransferases

Asparagine-linked (N-linked) protein glycosylation is one of the most abundant types of post-translational modification, occurring in all domains of life. The central enzyme in N-linked glycosylation is the oligosaccharyltransferase (OST), which catalyzes the covalent attachment of preassembled glycans to specific asparagine residues in target proteins. Whereas in higher eukaryotes the OST is comprised of eight different membrane proteins of which the catalytic subunit is STT3, in kinetoplastids and prokaryotes the OST is a monomeric enzyme bearing homology to STT3. Given their relative simplicity, these single-subunit OSTs (ssOSTs) have emerged as important targets for mechanistic dissection of poorly understood aspects of N-glycosylation and at the same time hold great potential for the biosynthesis of custom glycoproteins. To take advantage of this utility, this chapter describes a multipronged approach for studying and engineering ssOSTs that integrates in vivo screening technology with in vitro characterization methods, thereby creating a versatile and readily-adaptable pipeline for virtually any ssOST of interest.\n\nAbbreviations

synthetic biology↗

bioRxiv2017

A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases

Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 440 {micro}g/mL for the single-subunit OST enzyme, Protein glycosylation B (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life.

synthetic biology↗