Fn3 library construction
Oligonucleotides were purchased from MWG Biotech (High Point, NC, USA) and IDT DNA Technologies (Coralville, IA, USA). The NNB library was previously constructed as described (Hackel et al., 2008
). In the NNB library, the oligonucleotides encoding the BC, DE and FG loops were replaced by NNB codons; specifically, the DNA encoding amino acids 23–30 (DAPAVTVR), 52–56 (GSKST) and 77–86 (GRGDSPASSK) were replaced with (NNB)x
= 6, 7, 8 or 9 for the BC loop, x
= 4, 5, 6 or 7 for the DE loop and x
= 5, 6, 8 or 10 for the FG loop. The YS library replaced the DNA encoding amino acids 23–31 (DAPAVTVRY) and 77–86 with TMY codons that encode for serine and tyrosine in equal frequency. Loop lengths of 7, 8, 9 or 10 in the BC loop and 6, 7, 8 or 10 in the FG loop were included. Earlier selections from the NNB library rarely yielded FG loops of five amino acids, thus the YS library was constructed with a seven amino acid option rather than five. The DNA encoding amino acids 52–56 was replaced by a set of biased codons designed to yield 50% wild-type amino acid at G52, S53, S55 and T56. K54, because of its potential for steric and electrostatic hindrance of binding, was replaced by NNBx
= 0, 1 or 3. The degenerate portion of the oligonucleotide was ggBtcBNNBtcBacB where a, c, g and t represent mixtures of 70% of the indicated nucleotide and 10% of each of the other three. Full Fn3 genes were constructed by sequential annealing and extension of eight overlapping oligonucleotides. A 50 μl reaction was prepared with 0.2 μM oligonucleotide A (a2, b3, c6 or d7), 0.4 μM oligonucleotide B (a1, b4, c5 or d8), 1× polymerase buffer, 0.2 mM deoxynucleotide triphosphates, 1 mM MgSO4
, 1 U KOD HotStart DNA Polymerase (Novagen, Madison, WI, USA), 1 M betaine and 3% dimethyl sulfoxide. The mixture was denatured at 95° for 2 min, cycled 10 times through 94° for 30 s, 58° for 30 s and 68° for 1 min, and finally extended at 68° for 10 min. Forty microliters of products (a1 + a2, b3 + b4, c5 + c6, d7 + d8) were combined and thermally cycled at identical conditions. The appropriate strand (sense for a1 + a2 + b3 + b4 and anti-sense for c5 + c6 + d7 + d8) was amplified with 0.4 μM primer (p1 or p8) in a 100 µl reaction. The products were combined and thermally cycled under identical conditions. Full Fn3 genes were purified on an agarose gel, amplified by p1 and p8 in 100 μl reactions and concentrated with PelletPaint (Novagen). The plasmid acceptor vector pCTf1f4 (Lipovsek et al., 2007)
was digested with NcoI, NdeI and SmaI (New England Biolabs, Ipswich, MA, USA). Multiple aliquots of ~10 μg of Fn3 gene and ~3 μg of plasmid vector were combined with 50–100 μl of electrocompetent EBY100 and electroporated at 0.54 kV and 25 μF. Homologous recombination of the linearized vector and degenerate insert yielded intact plasmid. Cells were grown in YPD (10 g/l yeast extract, 20 g/l peptone, 20 g/l glucose) for 1 h at 30°, 250 rpm. The total number of transformants was determined by serial dilution plating on SD-CAA plates (0.1 M sodium phosphate, pH 6.0, 182 g/l sorbitol, 6.7 g/l yeast nitrogen base, 5 g/l casamino acids, 20 g/l glucose). The library was propagated in SD-CAA, pH 5.3 (0.07 M sodium citrate pH 5.3, 6.7 g/l yeast nitrogen base, 5 g/l casamino acids, 20 g/l glucose, 0.1 g/l kanamycin, 100 kU/l penicillin and 0.1 g/l streptomycin) at 30°, 250 rpm.
Binder selection and affinity maturation
Yeasts were grown in SD-CAA at 30°, 250 rpm to logarithmic phase, pelleted and resuspended to 1 × 107 cells/ml in SG-CAA (0.1 M sodium phosphate, pH 6.0, 6.7 g/l yeast nitrogen base, 5 g/l casamino acids, 19 g/l galactose, 1 g/l glucose, 0.1 g/l kanamycin, 100 kU/l penicillin and 0.1 g/l streptomycin) to induce protein expression. Induced cells were grown at 30°, 250 rpm for 8–24 h.
Magnetic bead sorts consisted of a negative selection for clones that do not bind streptavidin-coated beads followed by a positive selection for clones that bind biotinylated IgG complexed to streptavidin-coated beads as described (Ackerman et al., 2009
). Biotinylated goat or rabbit IgG 0.75 μg (Rockland Immunochemicals, Gilbertsville, PA, USA) was added to 4 × 106
streptavidin-coated magnetic Dynabeads (Invitrogen, Carlsbad, CA, USA) in 1 ml PBSA (0.01 M sodium phosphate, pH 7.4, 0.137 M NaCl, 1 g/l bovine serum albumin) and incubated at 4° for 12–24 h. Beads were washed using a Dynal magnet with PBSA. Yeasts displaying Fn3 were washed, resuspended in PBSA and incubated with 4 × 106
IgG-free streptavidin beads for 2–12 h at 4°. A magnet was applied to the cell/bead mixture and unbound cells were collected. The washed IgG-labeled beads were added to these cells and incubated at 4° for 2–12 h. The beads were applied to the magnet and washed with PBSA. The beads and attached cells were transferred to SD-CAA for growth.
The naïve library was sorted twice (zero washes at 4°, one wash at 4°) with growth and induction after each sort. The resultant population was labeled with 150 nM mouse anti-c-myc antibody (clone 9E10, Covance, Denver, PA, USA) followed by 25 nM goat anti-mouse phycoerythrin conjugate (Invitrogen). Full-length Fn3 clones, represented by cells with a positive phycoerythrin signal, were selected via FACS on an FACS Aria (Becton Dickinson, Franklin Lakes, NJ, USA) or MoFlo (Dako Cytomation, Carpinteria, CA, USA). Plasmid DNA was extracted and mutagenized as described (Hackel et al., 2008
). Error-prone PCR was performed on the full gene and each of the three loops; the mutated gene or shuffled combinations of the mutated loops were co-transformed with linearized plasmid vector to produce intact plasmid via homologous recombination. Transformed yeasts were grown in SD-CAA for further selection. The mutagenized population was sorted twice on magnetic beads (one wash at 4°, one wash at 22°) followed by c-myc+
FACS and further mutagenesis. After two magnetic bead sorts (one wash at 22°, two washes at 22°), binding to soluble IgG was assayed by flow cytometry. Yeast were labeled with 3.3 nM biotinylated IgG followed by 33 nM streptavidin–phycoerythrin conjugate. Cells with the highest phycoerythrin signal were collected by FACS and mutated. Remaining selections were performed with 20–500 pM biotinylated IgG and 67 nM chicken anti-c-myc followed by 150 nM streptavidin–fluorophore and 25 nM bovine anti-chicken phycoerythrin conjugate. Cells with the highest fluorophore:phycoerythrin ratio were selected by FACS.
DNA sequencing and point mutations
Multiple clones from several populations were sequenced. Plasmid DNA was isolated using the Zymoprep kit II (Zymo Research, Orange, CA, USA), cleaned using the Qiagen PCR Purification kit (Qiagen, Valencia, CA, USA) and transformed into DH5α (Invitrogen) or XL1-Blue E.coli (Stratagene, La Jolla, CA, USA). Individual clones were grown, miniprepped and sequenced using BigDye chemistry on an Applied Biosystems 3730.
Single amino acid mutations were introduced by standard site-directed mutagenesis using the QuikChange Mutagenesis Kit (Stratagene) according to the manufacturer's instructions. Clone construction was verified by DNA sequencing.
The plasmid for the clone of interest was transformed into yeast using the Frozen EZ Transformation Kit II (Zymo Research), and cells were grown and induced as for selection. Cells were washed in PBSA and resuspended in PBSA containing fluorophore-conjugated IgG over a range of concentrations. Sample volumes and cell densities were selected to ensure 10-fold excess of IgG relative to displayed Fn3. Samples were incubated at 22° for a sufficient time to ensure the approach to equilibrium was at least 98% complete. After incubation, cells were washed and analyzed on an FACS Calibur cytometer (Becton Dickinson). The relative binding was calculated by subtracting background signal, which was determined in an unlabeled control, and normalizing to the saturated signal at high concentrations. The equilibrium dissociation constant, Kd, was identified as the concentration corresponding to half-maximal binding.
The yeast surface display thermal denaturation assay (Orr et al., 2003)
was performed as described (Hackel et al., 2008
). Yeasts displaying the clone of interest were washed and resuspended in PBSA, incubated at 22–85° for 30 min and incubated on ice for 5 min. The cells were incubated in 20 nM fluorescein-conjugated IgG for 30 min, washed and analyzed on an Epics XL flow cytometer. The minimum and maximum fluorescence, the midpoint of thermal denaturation (T1/2
) and the enthalpy of unfolding at T1/2
were determined by minimizing the sum of squared errors between experimental data and theoretical values according to a two-state unfolding equation.
Yeasts displaying the clone of interest were incubated with 100 nM bovine IgG-phycoerythrin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), AlexaFluor488 conjugates of streptavidin, chicken IgG or mouse IgG (Invitrogen) or fluorescein conjugates of goat IgG, human IgG or rabbit IgG (Sigma). Cells were incubated for 30 min, washed with PBSA and analyzed by flow cytometry. To test lysozyme binding, cells were incubated with 100 nM biotinylated lysozyme for 30 min, washed and resuspended in 25 nM AlexaFluor488-conjugated streptavidin. Cells were washed and analyzed by flow cytometry. Fluorophore signal was compared with both unlabeled and non-displaying cells.
The Fn3 gene was digested with NheI and BamHI and transformed to a pET vector containing a HHHHHHKGSGK-encoding C-terminus. The six histidines enable metal affinity purification, and the pentapeptide provides two additional amines for chemical conjugation. The plasmid was transformed into Rosetta (DE3) E.coli (Novagen), which was grown in LB medium with 100 mg/l kanamycin and 34 mg/l chloramphenicol at 37°. Two hundred microliters of overnight culture were added to 100 ml of LB medium, grown to an optical density of 0.5 units, and induced with 0.5 mM IPTG overnight. Cells were pelleted, resuspended in lysis buffer [50 mM sodium phosphate, pH 8.0, 0.5 M NaCl, 5% glycerol, 5 mM CHAPS, 25 mM imidazole and 1× complete EDTA-free protease inhibitor cocktail (Roche, Indianapolis, IN, USA)] and exposed to four freeze-thaw cycles. The soluble fraction was clarified by centrifugation at 15 000g for 10 min and purified by metal affinity chromatography on TALON resin (Clontech, Mountain View, CA, USA).
Purified Fn3 (gI2.5.3T88I and rI4.5.5K27S/K56S) was biotinylated using EZ-Link Sulfo-N-hydroxysuccinimide-LC-biotin (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Excess biotin was removed using a Zeba desalting spin column (Pierce). Biotinylated Fn3 was added to 1 ml of strepavidin–agarose (Pierce) in a column and washed. Goat or rabbit serum was added to the column and flowthrough was reapplied once. The column was washed with three 5 ml aliquots of PBS. Protein was eluted with 0.1 M glycine, pH 2.5. The original serum, flowthrough, washes and elution were separated by SDS–PAGE on 12% BisTris gel (Invitrogen) in the absence of dithiothreitol. The gel was stained with SimplyBlue SafeStain (Invitrogen) and imaged.
Purified Fn3 (gI2.5.3T88I and rI4.5.5K27S/K56S) was labeled with DyLight633 N-hydroxysuccinimide-ester (Pierce) according to the manufacturer's instructions. Unreacted dye was removed using a Zeba desalting spin column. Yeasts were induced to display an irrelevant Fn3 clone with the HA and c-myc epitopes. As in all yeast surface display, a fraction of this population does not display any Fn3 as a result of plasmid loss. These cells serve as an internal negative control. One million yeasts were incubated with 50 nM anti-HA goat IgG or anti-c-myc rabbit IgG (Genscript, Piscataway, NJ, USA), washed and incubated with 50 nM DyLight633-conjugated Fn3. Cells were washed and analyzed on an FACS Calibur cytometer.