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Development of β-­lactamase based surface display systems in Saccharomyces cerevisiae for testing the effects of mutation in protein glycosylation on surface display efficiency (CROSBI ID 704810)

Prilog sa skupa u časopisu | sažetak izlaganja sa skupa

Lozančić, Mateja ; Žunar, Bojan ; Teparić, Renata ; Mrša, Vladimir Development of β-­lactamase based surface display systems in Saccharomyces cerevisiae for testing the effects of mutation in protein glycosylation on surface display efficiency // FEBS Open Bio. 2021. str. 254-254 doi: 10.1002/2211-5463.13205

Podaci o odgovornosti

Lozančić, Mateja ; Žunar, Bojan ; Teparić, Renata ; Mrša, Vladimir

engleski

Development of β-­lactamase based surface display systems in Saccharomyces cerevisiae for testing the effects of mutation in protein glycosylation on surface display efficiency

Surface display in yeast represents a valuable alternative to enzyme immobilization on solid surfaces as it is usually less complicated, less time-consuming, and eliminates the need for expensive enzyme purification. Instead, it provides constant production of recombinant protein composed of enzyme of interest fused with fragments of native yeast cell wall proteins. This way modified cells can be used in production processes for longer time compared to the purified enzymes. However, major disadvantages of these technique are low surface display efficiency and possible hyperglycosylation of recombinant proteins. By lowering the amount of glycosylation in cells, several effects are expected. Decreased glycosylation level of enzyme of interest could result in less disturbance of its conformation. It also causes a lesser density of the outer mannan layer of the cell wall, possibly increasing the availability of the substrate. To test the effects of inactivation of genes involved in O-glycosylation (genes from PMT group) and N-glycosylation (OCH1 and genes from MMN group) on the enzyme activity and surface display efficiency, two different systems using β-lactamase as a reporter were developed. In one, bla gene coding for β-lactamase was fused with HSP150 gene coding for cell wall protein resulting in recombinant protein covalently immobilized on the wall through linkage on its N- terminal end. Other system consists of bla gene fused with a fragment of CCW12 gene coding for its signalling sequence for binding of C-terminal part of protein onto GPI anchor. Immobilization can cause differences in protein folding, possibly leading to changes in enzyme activity. To reduce interference of this effect with observed changes in enzyme activity in mutant strains, we used different immobilization approach described earlier. Protein amount in the cell wall is assessed by measuring β-lactamase activity using nitrocefin as substrate and in a semi-quantitative manner by western blot.

Surface display, β-lactamase, Ccw12, Pir2, glycosylation

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Podaci o prilogu

254-254.

2021.

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objavljeno

10.1002/2211-5463.13205

Podaci o matičnoj publikaciji

FEBS Open Bio

FEBS Press

2211-5463

Podaci o skupu

45th FEBS Congress: Molecules of Life: Towards New Horizons (FEBS 2021)

poster

03.07.2021-08.07.2021

Ljubljana, Slovenija

Povezanost rada

Biotehnologija

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