TY - JOUR
T1 - Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
AU - Case, Brett A.
AU - Hackel, Benjamin J.
N1 - Publisher Copyright:
© 2016 Wiley Periodicals, Inc.
PY - 2016/8/1
Y1 - 2016/8/1
N2 - Protein ligand charge can impact physiological delivery with charge reduction often benefiting performance. Yet neutralizing mutations can be detrimental to protein function. Herein, three approaches are evaluated to introduce charged-to-neutral mutations of three cations and three anions within an affibody engineered to bind epidermal growth factor receptor. These approaches—combinatorial library sorting or consensus design, based on natural homologs or library-sorted mutants—are used to identify mutations with favorable affinity, stability, and recombinant yield. Consensus design, based on 942 affibody homologs, yielded a mutant of modest function (Kd = 11 ±4 nM, Tm = 62°C, and yield = 4.0 ± 0.8 mg/L as compared to 5.3 ± 1.7 nM, 71°C, and 3.5 ± 0.3 mg/L for the parental affibody). Extension of consensus design to 10 additional mutants exhibited varied performance including a substantially improved mutant (Kd = 6.9 ± 1.4 nM, Tm = 71°C, and 12.7 ± 0.9 mg/L yield). Sorting a homolog-based combinatorial library of 7 × 105 mutants generated a distribution of mutants with lower stability and yield, but did identify one strongly binding variant (Kd = 1.2 ± 0.3 nM, Tm = 69°C, and 6.0 ± 0.4 mg/L yield). Synthetic consensus design, based on the amino acid distribution in functional library mutants, yielded higher affinities (P = 0.05) with comparable stabilities and yields. The best of four analyzed clones had Kd = 1.7 ± 0.5 nM, Tm = 68°C, and 7.0 ± 0.5 mg/L yield. While all three approaches were effective in creating targeted affibodies with six charged-to-neutral mutations, synthetic consensus design proved to be the most robust. Synthetic consensus design provides a valuable tool for ligand engineering, particularly in the context of charge manipulation. Biotechnol. Bioeng. 2016;113: 1628–1638.
AB - Protein ligand charge can impact physiological delivery with charge reduction often benefiting performance. Yet neutralizing mutations can be detrimental to protein function. Herein, three approaches are evaluated to introduce charged-to-neutral mutations of three cations and three anions within an affibody engineered to bind epidermal growth factor receptor. These approaches—combinatorial library sorting or consensus design, based on natural homologs or library-sorted mutants—are used to identify mutations with favorable affinity, stability, and recombinant yield. Consensus design, based on 942 affibody homologs, yielded a mutant of modest function (Kd = 11 ±4 nM, Tm = 62°C, and yield = 4.0 ± 0.8 mg/L as compared to 5.3 ± 1.7 nM, 71°C, and 3.5 ± 0.3 mg/L for the parental affibody). Extension of consensus design to 10 additional mutants exhibited varied performance including a substantially improved mutant (Kd = 6.9 ± 1.4 nM, Tm = 71°C, and 12.7 ± 0.9 mg/L yield). Sorting a homolog-based combinatorial library of 7 × 105 mutants generated a distribution of mutants with lower stability and yield, but did identify one strongly binding variant (Kd = 1.2 ± 0.3 nM, Tm = 69°C, and 6.0 ± 0.4 mg/L yield). Synthetic consensus design, based on the amino acid distribution in functional library mutants, yielded higher affinities (P = 0.05) with comparable stabilities and yields. The best of four analyzed clones had Kd = 1.7 ± 0.5 nM, Tm = 68°C, and 7.0 ± 0.5 mg/L yield. While all three approaches were effective in creating targeted affibodies with six charged-to-neutral mutations, synthetic consensus design proved to be the most robust. Synthetic consensus design provides a valuable tool for ligand engineering, particularly in the context of charge manipulation. Biotechnol. Bioeng. 2016;113: 1628–1638.
KW - affibody
KW - charge
KW - consensus design
KW - epidermal growth factor receptor
UR - http://www.scopus.com/inward/record.url?scp=84977103662&partnerID=8YFLogxK
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U2 - 10.1002/bit.25931
DO - 10.1002/bit.25931
M3 - Article
C2 - 26724421
AN - SCOPUS:84977103662
SN - 0006-3592
VL - 113
SP - 1628
EP - 1638
JO - Biotechnology and bioengineering
JF - Biotechnology and bioengineering
IS - 8
ER -