Facile generation of antibody heavy and light chain diversities for yeast surface display by Golden Gate Cloning

2019 ◽  
Vol 400 (3) ◽  
pp. 383-393 ◽  
Author(s):  
Lukas Roth ◽  
Julius Grzeschik ◽  
Steffen C. Hinz ◽  
Stefan Becker ◽  
Lars Toleikis ◽  
...  
Keyword(s):  
Light Chain ◽  
Surface Display ◽  
Combinatorial Libraries ◽  
Yeast Surface Display ◽  
Golden Gate ◽  
Yeast Display ◽  
Step Method ◽  
One Step ◽  
Yeast Surface ◽  
Golden Gate Cloning

Abstract Antibodies can be successfully engineered and isolated by yeast or phage display of combinatorial libraries. Still, generation of libraries comprising heavy chain as well as light chain diversities is a cumbersome process involving multiple steps. Within this study, we set out to compare the output of yeast display screening of antibody Fab libraries from immunized rodents that were generated by Golden Gate Cloning (GGC) with the conventional three-step method of individual heavy- and light-chain sub-library construction followed by chain combination via yeast mating (YM). We demonstrate that the GGC-based one-step process delivers libraries and antibodies from heavy- and light-chain diversities with similar quality to the traditional method while being significantly less complex and faster. Additionally, we show that this method can also be used to successfully screen and isolate chimeric chicken/human antibodies following avian immunization.

scholarly journals Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands

2021 ◽  
Vol 22 (4) ◽  
pp. 1634
Author(s):  
John Bowen ◽  
John Schneible ◽  
Kaitlyn Bacon ◽  
Collin Labar ◽  
Stefano Menegatti ◽  
...  
Keyword(s):  
Cyclic Peptide ◽  
Surface Display ◽  
Combinatorial Libraries ◽  
Yeast Surface Display ◽  
Yeast Display ◽  
Ww Domains ◽  
Tev Protease ◽  
Peptide Modification ◽  
Modified Peptides ◽  
Alternative Routes

We present the construction and screening of yeast display libraries of post-translationally modified peptides wherein site-selective enzymatic treatment of linear peptides is achieved using bacterial transglutaminase. To this end, we developed two alternative routes, namely (i) yeast display of linear peptides followed by treatment with recombinant transglutaminase in solution; or (ii) intracellular co-expression of linear peptides and transglutaminase to achieve peptide modification in the endoplasmic reticulum prior to yeast surface display. The efficiency of peptide modification was evaluated via orthogonal detection of epitope tags integrated in the yeast-displayed peptides by flow cytometry, and via comparative cleavage of putative cyclic vs. linear peptides by tobacco etch virus (TEV) protease. Subsequently, yeast display libraries of transglutaminase-treated peptides were screened to isolate binders to the N-terminal region of the Yes-Associated Protein (YAP) and its WW domains using magnetic selection and fluorescence activated cell sorting (FACS). The identified peptide cyclo[E-LYLAYPAH-K] featured a KD of 1.75 μM for YAP and 0.68 μM for the WW domains of YAP as well as high binding selectivity against albumin and lysozyme. These results demonstrate the usefulness of enzyme-mediated cyclization in screening combinatorial libraries to identify cyclic peptide binders.

scholarly journals An overview of yeast cell wall proteins and their contribution in yeast display system

2020 ◽  
Vol 5 (4) ◽  
pp. 246-257
Author(s):  
SK Amir Hossain ◽  
SM Rifat Rahman ◽  
Toufiq Ahmed ◽  
Chanchal Mandal
Keyword(s):  
Cell Wall ◽  
Yeast Cell ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Yeast Cell Wall ◽  
Cell Wall Proteins ◽  
Display System ◽  
Yeast Display ◽  
Yeast Surface ◽  
Anchor Proteins

Yeast surface display has become an increasingly popular tool for protein engineering and library screening applications. Although, recent advances have greatly expanded the capability of yeast surface display, the protein display system is still far away from industrial application. One of the major components of a stable, efficient and successful yeast surface display system is cell wall anchor protein with which our desired foreign protein will be attached. We studied 80 different yeast cell wall anchored proteins originated mostly from Saccharomyces cerevisiae and Candida albicans. We studied in details all the cell wall proteins in order to find out suitable cell wall proteins to recommend for the researchers to use in the construction of yeast display system. We considered selective physical properties of different yeast cell wall proteins that are crucial for selecting best suited cell surface anchor proteins which are molecular weight, binding domain of anchor protein, length of amino acid and fusion site. Finally, our studies showed that Ccw11, Ccw12. Cwp1, Cwp2, Dan1, Gas1, Gas5, Exg1, Ycr89, Ecm33, Pga4, Sap9, Sap10, Pst1, Pir1, Pir2, Pir3, Pir4, Cis1, Scw4, Scw6, Bgl2, Uth1, Scw1 are the promising and suitable cell wall anchor proteins could be used in construction of yeast cell surface display system. Additionally, this review presents detailed information about all the cell wall proteins in a single work. The future researchers in this field will be able to construct more efficient yeast display system for recombinant protein production at industrial scale using the knowledge presented in this work. Asian J. Med. Biol. Res. June 2019, 5(4): 246-257

scholarly journals A novel one-step approach for the construction of yeast surface display Fab antibody libraries

2018 ◽  
Vol 17 (1) ◽  
Author(s):  
Simon Rosowski ◽  
Stefan Becker ◽  
Lars Toleikis ◽  
Bernhard Valldorf ◽  
Julius Grzeschik ◽  
...  
Keyword(s):  
Surface Display ◽  
Yeast Surface Display ◽  
Antibody Libraries ◽  
One Step ◽  
Yeast Surface

scholarly journals Isolation of Chemically Cyclized Peptide Binders Using Yeast Surface Display

2020 ◽  
Author(s):  
Kaitlyn Bacon ◽  
Abigail Blain ◽  
Matthew Burroughs ◽  
Nikki McArthur ◽  
Balaji M. Rao ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Cyclic Peptides ◽  
Cyclic Peptide ◽  
Surface Display ◽  
Combinatorial Libraries ◽  
Yeast Surface Display ◽  
Peptide Sequence ◽  
Interleukin 17 ◽  
Isolation And Characterization ◽  
Yeast Surface

AbstractCyclic peptides with engineered protein-binding activity have gained increasing attention for use in therapeutic and biotechnology applications. We describe the efficient isolation and characterization of cyclic peptide binders from genetically encoded combinatorial libraries using yeast surface display. Here, peptide cyclization is achieved by disuccinimidyl glutarate-mediated crosslinking of amine groups within a linear peptide sequence that is expressed as a yeast cell surface fusion. Using this approach, we first screened a library of cyclic heptapeptides by magnetic selection and fluorescence activated cell sorting (FACS), to isolate binders for a model target (lysozyme) with low micromolar binding affinity (KD ~ 1.2 - 3.7 µM). The isolated peptides bound lysozyme selectively, and only when cyclized. Importantly, we showed that yeast surface displayed cyclic peptides could be used to efficiently obtain quantitative estimates of binding affinity, without chemical synthesis of the selected peptides. Subsequently, to demonstrate broader applicability of our approach, we isolated cyclic heptapeptides that bind human interleukin-17 (IL-17) using yeast-displayed IL-17 as a target for magnetic selection, followed by FACS using recombinant IL-17. Molecular docking simulations and follow-up experimental analyses identified a candidate cyclic peptide that binds IL-17 in its receptor binding region with moderate affinity (KD ~ 300 nM). Taken together, our results show that yeast surface display can be used to efficiently isolate and characterize cyclic peptides generated by chemical modification from combinatorial libraries.

scholarly journals Screening of yeast display libraries of enzymatically-cyclized peptides to discover macrocyclic peptide ligands

2020 ◽  
Author(s):  
John Bowen ◽  
John Schneible ◽  
Collin Labar ◽  
Stefano Menegatti ◽  
Balaji M. Rao
Keyword(s):  
Cyclic Peptides ◽  
Cyclic Peptide ◽  
Surface Display ◽  
Peptide Ligands ◽  
Yeast Surface Display ◽  
Yeast Display ◽  
Ww Domains ◽  
Tev Protease ◽  
Yeast Surface ◽  
Alternative Routes

AbstractWe present the construction and screening of yeast display libraries of cyclic peptides wherein site-selective enzymatic cyclization of linear peptides is achieved using bacterial transglu-taminase. To this end, we developed two alternative routes, namely (i) yeast display of linear peptides followed by treatment with recombinant transglutaminase in solution; or (ii) intracellular co-expression of linear peptides and transglutaminase to achieve cyclization in the endoplasmic reticulum prior to yeast surface display. The cyclization yield was evaluated via orthogonal detection of epitope tags integrated in the yeast-displayed peptides by flow cytometry, and via comparative cleavage of cyclic vs. linear peptides by tobacco etch virus (TEV) protease. Subsequently, yeast display libraries of transglutaminase-cyclized peptides were screened to isolate binders to the N-terminal region of the Yes-Associated Protein (YAP) and its WW domains using magnetic selection and fluorescence activated cell sorting (FACS). The identified cyclic peptide cyclo[E-LYLAYPAH-K] featured a KD of 1.67 µM for YAP and 0.84 µM for WW as well as high binding selectivity against albumin and lysozyme. These results demonstrate the usefulness of yeast surface display for screening transglutaminase-cyclized peptide libraries, and efficient identification of cyclic peptide ligands.

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.

Sign in / Sign up

Export Citation Format

Share Document