Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor.

ABSTRACT: 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?×?10(5) 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. © 2016 Wiley Periodicals, Inc.

SUBMITTER: Case BA 

PROVIDER: S-EPMC5200887 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature