Affinity tuning of antibodies using yeast surface display
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Transcript of Affinity tuning of antibodies using yeast surface display
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Affinity tuning of antibodies using yeast surface display
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Yeast surface display of I-Ani1: Two epitope tags, One “antigen” binding site
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Yeast surface display of I-Ani1: single bp resolution of binding specificity
1 bp change 2 bp change 3 bp change
1 bp change 2 bp change
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Strategies for generating variants & libraries
Rational & Computational
Design
Error Prone PCR Combinatorial Libraries & Gene
Shuffling
Jim, Summer, Justin, Audrey, Umut , and
others?
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Optimal specific S/N ratio at physiological osmolarity
75 mM KCl 100 mM KCl 125 mM KCl 150 mM KCl 175 mM KCl 200 mM KCl341 mOsm 472 mOsm405 mOsm316 mOsm262 mOsm222 mOsm
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200 250 300 350 400 450 500
Sign
al/N
ois
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mOsm
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Analysis of computationally designed I-Ani1 variant: K24N T29R E31L R72S
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JH&ST-Ani combinatorial Lib1.0
-ve MACS Fraction
+ve MACS Fraction
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Targeting STS3 and STS4 to generate a high-affinity I-Ani1 variant
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Kd = 65nM
Kd = 8 nMKd = 20 nM
ReoAni
STS3/4 Lib1.0.A2
Yeast surface display of I-Ani1: ionic effects on HE specificity
Standard conditions: 150mM KCl, 10mM NaCl, 5mM CaCl2, 10mM HEPES, 0.2% BSA, 0.1% Galactose, pH 7.5