Displaying Lipase B from Candida antarctica in Pichia pastoris Using the Yeast Surface Display Approach: Prospection of a New Anchor and Characterization of the Whole Cell Biocatalyst

Levan has wide applications in chemical, cosmetic, pharmaceutical and food industries. The free levansucrase is usually used in the biosynthesis of levan, but the poor reusability and low stability of free levansucrase have limited its large-scale use. To address this problem, the surface-displayed levansucrase in Saccharomyces cerevisiae were generated and evaluated in this study. The levansucrase from Zymomonas mobilis was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using a various yeast surface display platform. The N-terminal fusion partner is based on a-agglutinin, and the C-terminal one is Flo1p. The yield of levan produced by these two whole-cell biocatalysts reaches 26 g/L and 34 g/L in 24 h, respectively. Meanwhile, the stability of the surface-displayed levansucrases is significantly enhanced. After six reuses, these two biocatalysts retained over 50% and 60% of their initial activities, respectively. Furthermore, the molecular weight and polydispersity test of the products suggested that the whole-cell biocatalyst of levansucrase displayed by Flo1p has more potentials in the production of levan with low molecular weight which is critical in certain applications. In conclusion, our method not only enable the possibility to reuse the enzyme, but also improves the stability of the enzyme.

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Optimizing secretion efficiency of industrially relevant recombinant proteins in Pichia pastoris by means of yeast surface display and cytometric sorting

New Biotechnology ◽

10.1016/j.nbt.2014.05.931 ◽

2014 ◽

Vol 31 ◽

pp. S189

Author(s):

Lars Toellner ◽

Fabian Schneider ◽

Brigitte Gasser ◽

Diethard Mattanovich

Keyword(s):

Pichia Pastoris ◽

Recombinant Proteins ◽

Surface Display ◽

Yeast Surface Display ◽

Secretion Efficiency ◽

Yeast Surface

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Synthetic repertoires derived from convalescent COVID-19 patients enable discovery of SARS-CoV-2 neutralizing antibodies and a novel quaternary binding modality

10.1101/2021.04.07.438849 ◽

2021 ◽

Author(s):

Jimmy D Gollihar ◽

Jason S McLellan ◽

Daniel R Boutz ◽

Jule Goike ◽

Andrew Horton ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Surface Display ◽

Functional Characterization ◽

Yeast Surface Display ◽

Spike Protein ◽

Emerging Pathogens ◽

Effective Interventions ◽

Yeast Surface

The ongoing evolution of SARS-CoV-2 into more easily transmissible and infectious variants has sparked concern over the continued effectiveness of existing therapeutic antibodies and vaccines. Hence, together with increased genomic surveillance, methods to rapidly develop and assess effective interventions are critically needed. Here we report the discovery of SARS-CoV-2 neutralizing antibodies isolated from COVID-19 patients using a high-throughput platform. Antibodies were identified from unpaired donor B-cell and serum repertoires using yeast surface display, proteomics, and public light chain screening. Cryo-EM and functional characterization of the antibodies identified N3-1, an antibody that binds avidly (Kd,app = 68 pM) to the receptor binding domain (RBD) of the spike protein and robustly neutralizes the virus in vitro. This antibody likely binds all three RBDs of the trimeric spike protein with a single IgG. Importantly, N3-1 equivalently binds spike proteins from emerging SARS-CoV-2 variants of concern, neutralizes UK variant B.1.1.7, and binds SARS-CoV spike with nanomolar affinity. Taken together, the strategies described herein will prove broadly applicable in interrogating adaptive immunity and developing rapid response biological countermeasures to emerging pathogens.

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