title: 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.
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota-Twin Cities, 421 Washington Avenue SE, 356 Amundson Hall, Minneapolis, Minnesota, 55455.
authors.forename: Brett A
authors.initials: BA
authors.lastname: Case
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota-Twin Cities, 421 Washington Avenue SE, 356 Amundson Hall, Minneapolis, Minnesota, 55455. hackel@umn.edu.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Hackel
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D000906:Antibodies / Q000737:chemistry* / Q000235:genetics; D016384:Consensus Sequence / Q000235:genetics*; D066246:ErbB Receptors / Q000737:chemistry* / Q000235:genetics; D009154:Mutation / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D000596:Amino Acids; D000906:Antibodies; D011993:Recombinant Fusion Proteins; D066246:ErbB Receptors
keywords: affibody; charge; consensus design; epidermal growth factor receptor