Engineering of Yeast Glycoprotein Expression

CRISPR-based peptide library display and programmable microarray self-assembly for rapid quantitative protein binding assays

“…In the future, CRISPR-based protein display and PICASSO may also be developed for classes of proteins that are typically incompatible with other displays. Engineered heterologous systems (Barber et al, 2018;Pirman et al, 2015;De Wachter et al, 2018) or eukaryotic cell lines could be used for dCas9-fusion library expression to best display full-length proteins with complex folding or posttranslational modifications accessible only in higher organisms. Additionally, PICASSO could be modified to colocalize multiple peptide or protein species to the same PICASSO microarrays could also be designed to contain synthetic antibody collections fused to dCas9, enabling multiplexed antigen detection via miniaturized parallel sandwich ELISAs.…”

What makes Komagataella phaffii non-conventional?

“…( 2013 ), highlighting some noteworthy differences between the different species. One aspect that is specifically relevant to the production of biopharmaceuticals are differences in N- and O-glycosylation, as both types of PTM have been demonstrated to affect pharmacokinetics and pharmacodynamics of recombinantly produced proteins (De Wachter, Van Landuyt and Callewaert 2018 ; Zhou and Qiu 2019 ). N-glycosylation plays a very important role in the folding and quality control (QC) process of glycosylated proteins.…”

“…Subsequently, the terminal α-1,2 and α-1,3 glucose residues are removed by respective glucosidases and one α-1,2-mannose is removed by an ER-residing α-1,2-mannosidase, resulting in an Man 8 GlcNAc 2 glycan. Further N-glycan modifications of properly folded proteins take place in the Golgi apparatus, where yeasts add mannose and mannosylphosphate sugars to the Man 8 GlcNAc 2 glycan core, generating N-glycans of the high-mannose type (Hamilton and Gerngross 2007 ; De Wachter, Van Landuyt and Callewaert 2018 ). N-glycan structure and side chain composition can differ substantially between yeast species (Thak et al .…”

A Perspective on Synthetic Biology in Drug Discovery and Development—Current Impact and Future Opportunities

“…Yeast has the advantage to be able to efficiently secrete and display proteins and can be engineered to conduct posttranslational modifications like glycosylation similar to mammalian systems. [74][75][76][77] In contrast to bacterial hosts like E. coli, S. cerevisiae has intracellular organelle systems, including extended membrane structures like the endoplasmic reticulum and Golgi apparatus. These are important to functionally express certain enzymes, like cytochrome P450 monooxygenases, which are essential for the biosynthesis of many natural products of high structural diversity.…”