Yeast Surface Display for Antibody Isolation: Library Construction, Library Screening, and Affinity Maturation

2014 ◽  
pp. 151-181 ◽  
Author(s):  
James A. Van Deventer ◽  
Karl Dane Wittrup
Keyword(s):  
Surface Display ◽  
Affinity Maturation ◽  
Yeast Surface Display ◽  
Library Screening ◽  
Library Construction ◽  
Yeast Surface

scholarly journals Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries

2019 ◽  
Vol 2019 ◽  
pp. 1-22 ◽  
Author(s):  
Biancamaria Cembrola ◽  
Valentino Ruzza ◽  
Fulvia Troise ◽  
Maria Luisa Esposito ◽  
Emanuele Sasso ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Surface Display ◽  
Affinity Maturation ◽  
Yeast Surface Display ◽  
Antigen Concentration ◽  
Selection Scheme ◽  
High Affinity ◽  
Novel Approach ◽  
Yeast Surface

The affinity engineering is a key step to increase the efficacy of therapeutic monoclonal antibodies and yeast surface display is the most widely used and powerful affinity maturation approach, achieving picomolar binding affinities. In this study, we provide an optimization of the yeast surface display methodology, applied to the generation of potentially therapeutic high affinity antibodies targeting the immune checkpoint PD-L1. In this approach, we coupled a 10-cycle error-prone mutagenesis of heavy chain complementarity determining region 3 of an anti‐PD-L1 scFv, previously identified by phage display, with high-throughput sequencing, to generate scFv-yeast libraries with high mutant frequency and diversity. In addition, we set up a novel, faster and effective selection scheme by fluorescence-activated cell sorting, based on a fast drop of the antigen concentration between the first and the last selection cycles, unlike the gradual decrease typical of current selection protocols. In this way we isolated 6 enriched mutated scFv-yeast clones overall, showing an affinity improvement for soluble PD-L1 protein compared to the parental scFv. As a proof of the potency of the novel approach, we confirmed that the antibodies converted from all the mutated scFvs retained the affinity improvement. Remarkably, the best PD-L1 binder among them also bound with a higher affinity to PD-L1 expressed in its native conformation on human-activated lymphocytes, and it was able to stimulate lymphocyte proliferation in vitro more efficiently than its parental antibody. This optimized technology, besides the identification of a new potential checkpoint inhibitor, provides a tool for the quick isolation of high affinity binders.

scholarly journals Affinity maturation of human CD4 by yeast surface display and crystal structure of a CD4-HLA-DR1 complex

2011 ◽  
Vol 108 (38) ◽  
pp. 15960-15965 ◽  
Author(s):  
X. X. Wang ◽  
Y. Li ◽  
Y. Yin ◽  
M. Mo ◽  
Q. Wang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Surface Display ◽  
Affinity Maturation ◽  
Yeast Surface Display ◽  
Yeast Surface

scholarly journals Evaluation of the yeast surface display system for screening of functional nanobodies

AMB Express ◽  
2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kaho Kajiwara ◽  
Wataru Aoki ◽  
Mitsuyoshi Ueda
Keyword(s):  
Surface Display ◽  
Yeast Surface Display ◽  
Display System ◽  
Yeast Surface

scholarly journals Development and Characterization of Two Types of Surface Displayed Levansucrases for Levan Biosynthesis

Catalysts ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 757
Author(s):  
Huiyi Shang ◽  
Danni Yang ◽  
Dairong Qiao ◽  
Hui Xu ◽  
Yi Cao
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Large Scale ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Fusion Partner ◽  
Whole Cell ◽  
Food Industries ◽  
Yeast Surface ◽  
The Stability

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.

Yeast Surface Display in Combination with Fluorescence‐activated Cell Sorting Enables the Rapid Isolation of Antibody Fragments Derived from Immunized Chickens

2018 ◽  
Vol 14 (4) ◽  
pp. 1800466 ◽  
Author(s):  
Julius Grzeschik ◽  
Desislava Yanakieva ◽  
Lukas Roth ◽  
Simon Krah ◽  
Steffen C. Hinz ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Surface Display ◽  
Antibody Fragments ◽  
Yeast Surface Display ◽  
Fluorescence Activated Cell Sorting ◽  
Rapid Isolation ◽  
Yeast Surface
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