20.109(S20):Module 3
Contents
Module 3
Lecturer: Leslie McClain
Instructors: Noreen Lyell and Leslie McClain, and Becky Meyer
Research assistant: Sarah Cowles
TAs: Kevin Chung and Joe Kreitz
Lab manager: Hsinhwa Lee
Overview
An antibody is a soluble or membrane bound protein, produced by immune cells in blood. The purpose of an antibody is to recognize foreign substances, called antigens, and mark them for removal by cells that compromise our immune system. If one has a properly functioning immune system you've all been making new and improved antibodies your entire life.
Antibodies are also a critical reagent in medicine, research and diagnostics. Scientists have found ways to purify, modify and harvest antibodies from animals and to also engineer cells to produce specific antibodies. In Mod3 we'll use a method developed in the Wittrup lab to screen a yeast library of antibodies, against a particular antigen, using Fluorescence Assisted Cell Sorting, and try to identify a single antibody that shows improved antigen binding in a quantitative binding assay.
This module was developed thanks to the invaluable help and support of Wittrup lab PhD student, Sarah Cowles, and the generous contribution of reagents from Prof. Wittrup.
Lab links: day by day
M3D1: Enrich candidate clones using fluorescence-activated cell sorter (FACS)
M3D2: Harvest candidate clones and prepare for sequencing
M3D3: Identify clones to characterize
M3D4: Characterize clone by titration using flow cytometry
M3D5: Analyze titration curves
Assignments
Research proposal presentations
Mini-report
References
- Isolating and engineering human antibodies using yeast surface display. Nature Protocols. 1, p755–768(2006).
- Yeast Surface Display for Antibody Isolation: Library Construction, Library Screening, and Affinity Maturation. Methods in Molecular Biology . 1131:151-81(2014).
- Applications of Yeast Surface Display for Protein Engineering. Methods in Molecular Biology (2015) 1319:155-75.
- Examples of Using Yeast Surface Display for Non-antibody Proteins:
- Stability and CDR Composition Biases Enrich Binder Functionality Landscapes. J. Mol. Biol. (2010) 401, 84–96
- Beyond Epitope Binning: Directed in Vitro Selection of Complementary Pairs of Binding Proteins. ACS Comb. Sci. (2020) 22, 49−60.
- Protocol for expressing full antibodies in mammalian cells:Transient expression of human antibodies in mammalian cells. Nature Protocols (2018) 13(10), 99-117