Aprotinin and cytochrome c were used as molecular weight standards. Discussion Consensus design was successful in achieving comparable affinity (Kd = 11 4 nM for EA35 vs. (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. protein A (Nilsson (New England Biolabs), and proper transformants were selected on lysogeny broth (10 g/L tryptone, 5.0 g/L yeast extract, 10.0 g/L sodium chloride in water) plates (15 g/L agar) containing kanamycin (50 mg/L). Two mL of saturated culture was added to 100 mL of lysogeny broth medium in a 250 mL shake flask, incubated UNC 2400 at 37 C, 250 rpm until an optical density at 600 nm between 0.5 and UNC 2400 1 units was achieved and induced with 0.5 mM isopropyl -D-1-thiogalactopyranoside for 2 hours at 25 C 28 C. Cells were pelleted, resuspended in lysis buffer (50 mM sodium phosphate (pH 8.0), 0.5 M sodium chloride, 5% glycerol, 5 mM 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, and 25 mM imidazole), and underwent four freeze-thaw cycles. The soluble fraction was isolated from cell lysate via centrifugation at 12,000for 10 min. Affibodies were purified by immobilized metal affinity chromatography on 0.2 mL resin volume Ni-NTA HisPur spin columns (Thermo Fisher Scientific) and concentration assessed using absorbance at 280 nm on a Synergy H1 microplate reader (BioTek). Clones were further purified by reversed-phase high-performance liquid chromatography with a C18 column using a 15-minute gradient of 25% to 80% elution buffer (90% acetonitrile, 9.9% water, 0.1% trifluoroacetic acid) and the remaining solution composed of running buffer (99.9% water, 0.1% trifluoroacetic acid). Isolated affibodies were lyophilized. Yields are presented as the average standard error of eight or nine production runs. Library Construction and Sorting Genes for four charge-reduced affibody sub-libraries were constructed via overlap extension PCR of 12C15 oligonucleotides per sub-library, with degenerate codons present at six of the following positions: 2, 4, 7, 8, 15, 37, 49, 53, or 58 (Table S1). PCR reaction products and linearized pCT surface display vector were homologously recombined upon transformation via electroporation into EBY100 yeast (Chao (New England Biolabs) and grown on plates (lysogeny broth with 15 g/L agar and 50 mg/L kanamycin). Genes from fifty-four plate colonies were sequenced UNC 2400 and found to all encode for affibodies. Sixteen sequences had combinations of charge mutations not designed into the sub-libraries C in some cases more neutralizations and in other cases less C and were discarded. The remaining thirty-eight sequences were used to create synthetic consensus frequencies. Circular Dichroism Purified, lyophilized affibody clones were resuspended in PBS to a concentration of 0.5 C 1 g/L. Ellipticity was measured between 200 and 260 nm wavelengths at room temperature on a Jasco J-815 spectrophotometer in a quartz cuvette with 1 mm path length. Thermal denaturation was performed by measuring ellipticity at 220 nm from 20 to 98 C (2 C/min). The midpoint of thermal denaturation (Tm) Rabbit Polyclonal to CREBZF was calculated using a two-state UNC 2400 unfolding model. Heated samples were cooled to 20 C and subjected to a post-heating wavelength scan. Affinity Measurement A431 epidermoid carcinoma cells, which express approximately 2.8 106 EGFR per cell (Spangler (Friedman (Feldwisch positively.