Facile generation of antibody heavy and light chain diversities for yeast surface display by Golden Gate Cloning
-
Lukas Roth
, Julius Grzeschik , Steffen C. Hinz , Stefan Becker , Lars Toleikis , Michael Busch , Harald Kolmar , Simon Krahund Stefan Zielonka
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.
Acknowledgments
We are grateful to Ramona Gaa, Iris Willenbuecher, Kerstin Hallstein and Deniz Demir for experimental support.
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Supplementary Material
The online version of this article offers supplementary material (https://doi.org/10.1515/hsz-2018-0347).
©2019 Walter de Gruyter GmbH, Berlin/Boston
Artikel in diesem Heft
- Frontmatter
- Protein engineering comes of age
- Microbial transglutaminase for biotechnological and biomedical engineering
- Computational design of structured loops for new protein functions
- Formylglycine-generating enzymes for site-specific bioconjugation
- Chemical modification of neuropeptide Y for human Y1 receptor targeting in health and disease
- Manipulating the stereoselectivity of a thermostable alcohol dehydrogenase by directed evolution for efficient asymmetric synthesis of arylpropanols
- Radiometal-labeled anti-VCAM-1 nanobodies as molecular tracers for atherosclerosis – impact of radiochemistry on pharmacokinetics
- Hit evaluation of an α-helical peptide: Ala-scan, truncation and sidechain-to-sidechain macrocyclization of an RNA polymerase Inhibitor
- Highly flexible, IgG-shaped, trivalent antibodies effectively target tumor cells and induce T cell-mediated killing
- An engineered lipocalin that tightly complexes the plant poison colchicine for use as antidote and in bioanalytical applications
- Sequence selection by FitSS4ASR alleviates ancestral sequence reconstruction as exemplified for geranylgeranylglyceryl phosphate synthase
- Facile generation of antibody heavy and light chain diversities for yeast surface display by Golden Gate Cloning
- Peptide binding affinity redistributes preassembled repeat protein fragments
- Directed evolution of the 3C protease from coxsackievirus using a novel fluorescence-assisted intracellular method
- Rational design of an improved photo-activatable intein for the production of head-to-tail cyclized peptides
- Characterization and engineering of photoactivated adenylyl cyclases
Artikel in diesem Heft
- Frontmatter
- Protein engineering comes of age
- Microbial transglutaminase for biotechnological and biomedical engineering
- Computational design of structured loops for new protein functions
- Formylglycine-generating enzymes for site-specific bioconjugation
- Chemical modification of neuropeptide Y for human Y1 receptor targeting in health and disease
- Manipulating the stereoselectivity of a thermostable alcohol dehydrogenase by directed evolution for efficient asymmetric synthesis of arylpropanols
- Radiometal-labeled anti-VCAM-1 nanobodies as molecular tracers for atherosclerosis – impact of radiochemistry on pharmacokinetics
- Hit evaluation of an α-helical peptide: Ala-scan, truncation and sidechain-to-sidechain macrocyclization of an RNA polymerase Inhibitor
- Highly flexible, IgG-shaped, trivalent antibodies effectively target tumor cells and induce T cell-mediated killing
- An engineered lipocalin that tightly complexes the plant poison colchicine for use as antidote and in bioanalytical applications
- Sequence selection by FitSS4ASR alleviates ancestral sequence reconstruction as exemplified for geranylgeranylglyceryl phosphate synthase
- Facile generation of antibody heavy and light chain diversities for yeast surface display by Golden Gate Cloning
- Peptide binding affinity redistributes preassembled repeat protein fragments
- Directed evolution of the 3C protease from coxsackievirus using a novel fluorescence-assisted intracellular method
- Rational design of an improved photo-activatable intein for the production of head-to-tail cyclized peptides
- Characterization and engineering of photoactivated adenylyl cyclases