Comprehensive Saturation Mutagenesis Libraries

Comprehensive single site saturation mutagenesis (SSM) libraries were constructed using nicking mutagenesis exactly as described (Wren-beck et al., 2016 (link)). All mutagenic oligos were designed using Quik Change primer design Program (Agilent) and were ordered from IDT (Coralville, IA). For pro-cNGF, two separate libraries were prepared: library 1 covered residues Glu-102-Asn-52 and library 2 covered residues Ile-51-Arg0. For proΔ1,2-cNGF, the library covered residues Gln-55-Arg0. For conformational epitope mapping pro.v4-cNGF was split into two libraries, with library 1 covering residues Ser1-Asp60 and library 2 covering residues Pro61-Ala120. Library plasmid DNA was transformed into chemically competent S. cerevisiae EBY100, grown, and stored in yeast storage buffer at −80°C exactly according to published protocols (Medina-Cucurella & Whitehead, 2018 (link)).

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Medina-Cucurella A.V., Zhu Y., Bowen S.J., Bergeron L.M, & Whitehead T.A. (2018). Pro region engineering of nerve growth factor by deep mutational scanning enables a yeast platform for conformational epitope mapping of anti-NGF monoclonal antibodies. Biotechnology and bioengineering, 115(8), 1925-1937.

Publication 2018

Quik Change primer design Program

At protocols Buffer Library Mutagenic Oligos Plasmid Primer Wren Yeast

Corresponding Organization : Michigan State University

Other organizations : Zoetis (United States)

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Variable analysis

independent variables

Single site saturation mutagenesis (SSM) libraries for pro-cNGF, proΔ1,2-cNGF, and conformational epitope mapping pro.v4-cNGF

dependent variables

Not explicitly mentioned

control variables

Mutagenic oligos designed using Quik Change primer design Program (Agilent)
Mutagenic oligos ordered from IDT (Coralville, IA)
Library plasmid DNA transformed into chemically competent S. cerevisiae EBY100
Yeast cells grown and stored in yeast storage buffer at −80°C according to published protocols (Medina-Cucurella & Whitehead, 2018)

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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