Fn3 library construction
Oligonucleotides were purchased from MWG Biotech (High Point, NC) and Integrated DNA Technologies (Coralville, IA) (see Supplementary Data online for sequences). The Fn3 library was constructed to produce wild-type sequence in the framework regions and to randomize the BC,DE, and FG loops of Fn3. The DNA encoding for amino acids 23–30 (DAPAVTVR) was replaced by (NNB)x where x=6, 7, 8, or 9 to yield a loop length that is −2, −1, 0, or +1 amino acids relative to wild type. Similarly, the DNA for amino acids 52–56 (GSKST) was replaced by (NNB)y where y=4, 5, 6, or 7, and the DNA for amino acids 77–86 (GRGDSPASSK) was replaced by (NNB)z where z=5, 6, 8, or 10.
The library was constructed by sequential annealing and extension of eight overlapping oligonucleotides.6
The following components were combined in a 50-µL reaction: two oligonucleotides (0.2 µM a2, b3nmix
, c6t, or d7nmix
+ 0.4 µMa1, b4n, c5nmix
, or d8n, respectively), 1× polymerase buffer, 0.2mMdeoxynucleotide triphosphate (dNTP),1mM MgSO4
, 1 U KOD Hot Start DNA Polymerase (Novagen, Madison, WI), 1 M betaine, and 3% dimethyl sulfoxide. The mixture was denatured at 95 °C for 2 min followed by 10 cycles of 94 °C for 30 s, 58 °C for 30 s, and 68° for 1 min and a final extension of 68 °C for 10 min. Forty microliters of the products (a1+a2, b3nmix
+c6t, or d7nmix
+d8n) were combined and thermally cycled at identical conditions. Two microliters of this product was combined with 0.4 µM primer (a1–b4n amplified with p1; c5nmix
–d8n amplified with p8) in a new 100-µL reaction and thermally cycled under identical conditions to amplify the appropriate strand. The products were combined and thermally cycled at identical conditions. The final products were concentrated with PelletPaint (Novagen). The plasmid acceptor vector pCTf1f4 (Ref. 6) was digested with NcoI, NdeI, and SmaI (New England Biolabs, Ipswich, MA). Multiple aliquots of ~10 µg of Fn3 gene and 3 µg plasmid vector were combined with 50–100 µL of electrocompetent EBY100 and electroporated at 0.54 kV and 25µF. Homologous recombination of the linearized vector and degenerate insert yielded intact plasmid. Cells were grown in YPD (1% yeast extract, 2% peptone, 2% glucose) for 1 h at 30 °C, 250 rpm. The number of total transformants was 6.5×107
cells as determined by serial dilutions plated 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 by selective growth 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 °C, 250 rpm.
Fluorescence-activated cell sorting
Yeast were grown in SD-CAA, pH 5.3, at 30 °C, 250 rpm to logarithmic phase, pelleted, and resuspended to 1×107 cells/mL in SG-CAA, pH 6.0 (0.1M sodium phosphate, pH 6.0, 6.7 g/L yeast nitrogen base, 5 g/L casamino acids, 19 g/L dextrose, 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 °C, 250 rpm for 12–24h.
Round 0 (three FACS selections) and round 1 (two FACS selections) were conducted with multivalent lysozyme prepared by incubating streptavidin–fluorophore (R-phycoerythrin, AlexaFluor488, or AlexaFluor633; Invitrogen, Carlsbad, CA) with biotinylated lysozyme (Sigma, St. Louis, MO) in a 1:3 ratio in phosphate-buffered saline with bovine serum albumin (PBSA). Yeast were pelleted, washed in 1 mL PBSA (0.01 M sodium phosphate, pH 7.4, 0.137 M sodium chloride, 1 g/L bovine serum albumin), resuspended in PBSA with 10–40 mg/L mouse anti-c-myc antibody (clone 9E10, Covance), and incubated on ice. Cells were washed with 1 mL PBSA and resuspended in PBSA with multivalent lysozyme (0.5 µM for the first four sorts and 50 nM for the fifth sort) and goat antimouse antibody conjugated to R-phycoerythrin, Alexa-Fluor488, or AlexaFluor633.
Intermediate FACS selections were conducted with near-equilibrium labeling with monovalent lysozyme. Three, two, two, and three selections were performed in rounds 2–5, respectively. Yeast were pelleted, washed in 1 mL PBSA, resuspended in PBSA with biotinylated lysozyme (ranging from 1 µM to 20 pM) and mouse anti-c-myc antibody, and incubated on ice. Cells were then washed with 1 mL PBSA and resuspended in PBSA with streptavidin–fluorophore and fluorophore-conjugated goat anti-mouse antibody.
FACS selections of very high affinity populations were conducted with kinetic competition. Two, three, and two selections were performed in rounds 6–8. Yeast were washed and incubated briefly with 1–2 nM biotinylated lysozyme. Yeast were then washed and resuspended with PBSA with 140 nM unbiotinylated lysozyme (to prevent further association of labeled target) and incubated at room temperature for 2 h to 7 days to enable dissociation of biotinylated lysozyme. Cells were washed in PBSA, resuspended in PBSA with mouse anti-c-myc antibody, and incubated on ice. Cells were washed and labeled with secondary reagents as in equilibrium labeling.
In all cases, labeled cells were washed with 1 mL PBSA, resuspended in 0.5–2.0 mL PBSA and analyzed by flow cytometry using either a MoFlo (Cytomation) or Aria (Becton Dickinson) cytometer. c-myc
+ cells with the top 0.2–3% of lysozyme binding/c-myc display ratio were selected. Collected cells were grown in SD-CAA, pH 5.3, at 30 °C, 250 rpm and either induced in SG-CAA, pH 6.0, for further selection or used for plasmid recovery.
Plasmid DNA from 1×108
cells was isolated using two columns of Zymoprep kit II (Zymo Research, Orange, CA) according to the manufacturer’s instructions except for additional centrifugation of neutralized precipitate. The zymoprep elution was cleaned using the Qiagen PCR Purification kit (Qiagen, Valencia, CA), and eluted in 40 µL of elution buffer. Error-prone PCR of the entire Fn3 gene was performed in a 50-µL reaction containing 1×Taq
buffer, 2 mM MgCl2
, 0.5 µM each of primers W5 and W3, 0.2 mM (each) dNTPs, 5 µL of zymoprepped DNA template, 2 mM 8-oxo-deoxyguanosine triphosphate (TriLink, San Diego, CA), 2 mM 2′-deoxy-p-nucleoside-5′-triphosphate (Tri-Link), and 2.5 U of Taq
DNA polymerase (Invitrogen). In parallel, error-prone PCR of the loop regions was performed via three separate 50 µL reactions with 20 mM 8-oxo-deoxyguanosine triphosphate and 20 mM 2′-deoxy-p-nucleoside-5′-triphosphate and primers BC5 and BC3 for the BC loop, DE5 and DE3 for the DE loop, and FG5 and FG3 for the FG loop. The reaction mixtures were denatured at 94 °C for 3 min, cycled 15 times at 94 °C for 45 s, 60 °C for 30 s, and 72 °C for 90 s, and finally extended at 72 °C for 10 min. Multiple preliminary mutagenesis reactions of the wild-type plasmid were conducted at different nucleotide analog concentrations. Sequence analysis and comparison to a theoretical framework30
indicated the aforementioned conditions yield one to five amino acid mutations per gene. The PCR products were purified by agarose gel electrophoresis and each amplified in four 100-µL PCR reactions containing 1× Taq
buffer, 2 mMMgCl2
, 1 µM of each primer, 0.2 mM (each) dNTPs, 4 µL of error-prone PCR product (of 40 µL from gel extraction), and 2.5U of Taq
DNA polymerase. The reactions were thermally cycled at the same conditions except that 35 cycles were used. Reaction products were concentrated with PelletPaint (Novagen) and resuspended in 1 µL of water.
Plasmid pCT-Fn3 was digested with PstI, BtgI, and BamHI to create linearized vector pCT-Fn3-Gene with the entire Fn3 gene removed. Plasmid pCT-Fn3 was digested with BclI, BtgI, and PasI to create linearized vector pCT-Fn3-Loop with the wild-type gene removed from the BC loop through the FG loop.
Electrocompetent EBY100 (100 µL) was combined with 0.5–2.0 µg of pCT-Fn3-Gene and the gene-based PCR product and electroporated at 0.54 kV and 25 µF in a 2 mM electroporation cuvette. Similarly, 100 µL of electrocompetent EBY100 was combined with 0.5–2.0 µg of pCT-Fn3-Loop and the three loop-based PCR products and electroporated. Homologous recombination of the linearized vector and mutagenized insert(s) yielded intact plasmid. Cells were grown in YPD for 1 h at 30 °C, 250 rpm. The medium was switched to SD-CAA to enable selective propagation of successful transformants via growth at 30 °C, 250 rpm, for 24–48 h.
Plasmid DNA was isolated using the Zymoprep kit II, cleaned using the Qiagen PCR Purification kit, and transformed into DH5α (Invitrogen) or XL1-Blue E. coli (Stratagene, La Jolla, CA). Individual clones were grown, miniprepped, and sequenced using BigDye chemistry on an Applied Biosystems 3730.
Measurement of Kd, kon, and koff
The equilibrium dissociation constant for a clone was determined essentially as described.26
Briefly, yeast containing the plasmid for an Fn3 clone was grown and induced as for FACS selection. Cells were washed in 1 mL PBSA and resuspended in PBSA containing biotinylated lysozyme in concentrations generally spanning 4 orders of magnitude surrounding the equilibrium dissociation constant. The numbers of cells and sample volumes were selected to ensure excess lysozyme relative to Fn3. For clones of low picomolar affinity, this criterion necessitates very low cell density, which makes cell collection by centrifugation procedurally difficult. To obviate this difficulty, uninduced cells are added to the sample to enable effective cell pelleting during centrifugation with no effect on lysozyme binding of the Fn3-displaying induced cells. Cells were incubated at 25 °C for sufficient time to ensure that the approach to equilibrium was at least 98% complete. Cells were then pelleted, washed with 1 mL PBSA, and incubated in PBSA with 10 mg/L streptavidin–R-phycoerythrin for 10–30 min. Cells were washed and resuspended with PBSA and analyzed with an Epics XL flow cytometer. The minimum and maximum fluorescence and the Kd
value were determined by minimizing the sum of squared errors.
For determination of the dissociation constant, koff, clonal cell cultures were grown, induced, and washed as above. Cells were incubated in PBSA with a saturating concentration of biotinylated lysozyme at 25 °C. At various times, an aliquot of cells was washed with PBSA with excess unbiotinylated lysozyme, resuspended in PBSA with excess unbiotinylated lysozyme, and incubated at 25 °C. Simultaneously, all samples of differing dissociation times were washed with PBSA and incubated in 10 mg/L streptavidin–R-phycoerythrin for 10–30 min. Cells were washed and resuspended with PBSA and analyzed with an Epics XL flow cytometer. The minimum and maximum fluorescence and the koff value were determined by minimizing the sum of squared errors.
For determination of the association constants, kon, clonal cell cultures were grown, induced, and washed as above. At various times, an aliquot of cells was resuspended in biotinylated lysozyme and incubated at 25 °C. Simultaneously, all samples of differing association times were washed with PBSA with excess unbiotinylated lysozyme and incubated in PBSA with 10 mg/L streptavidin–R-phycoerythrin for 10–30 min. Cells were washed and resuspended with PBSA and analyzed with an Epics XL flow cytometer. The maximum fluorescence and kon were determined by minimizing the sum of squared errors assuming a 1:1 binding model. The experimentally determined value of koff was used to determine the effective association rate, kon[lysozyme]+koff.
The equilibrium dissociation constant was also determined for the soluble forms of L7.5.1 and Cons0.4.1 by equilibrium competition titration. Varying concentration of purified Fn3 domains were incubated with 20 pM biotiny-lated lysozyme in 50 mL of PBSA. Yeast displaying L7.5.1 were added and incubated for 7 days to near equilibrium. Cells were then pelleted, washed with 1 mL PBSA, and incubated in PBSA with 10 mg/L streptavidin–R-phycoerythrin for 15 min. Cells were washed and resuspended with PBSA and analyzed with an Epics XL flow cytometer. A two-state binding model was assumed and the minimum and maximum fluorescence and equilibrium dissociation constant were determined by minimizing the sum of squared errors.
Fn3 production and biophysical characterization
Fn3 clones were produced as previously described.6
Briefly, BL21(DE3)pLysS E. coli
(Invitrogen) containing the pET-24b-based Fn3 plasmid were grown in Luria–Bertani medium with 50 µg/mL kanamycin and 34 µg/mL chloramphenicol at 37 °C, 250 rpm, to an A600
of 0.1–0.2 and induced with 0.5 mM IPTG for 18–24 h at 30 °C, 250 rpm. Cells were lysed by sonication and the insoluble fraction was removed by centrifugation at 19,000g for 40 min. His6
-tagged Fn3 was purified from the soluble fraction with TALON Superflow Metal Affinity Resin (Clontech), dialyzed against PBS, and concentrated to 0.5 mL with an Amicon Ultra centrifugal filter (Millipore).
The oligomeric state was analyzed by size-exclusion chromatography on a Superdex 75 HR10/300 column (Amersham Pharmacia Biotech, Piscataway, NJ). Monomer was isolated for biophysical analysis. PBS standards or monomeric protein in PBS was thermally denatured from 25 to 95 °C at a rate of 1 °C/min in a differential scanning calorimeter (VP-DSC, MicroCal). Irreversible aggregation of L7.5.1 occurs at high temperatures. The midpoint of thermal denaturation for this clone is identified as the temperature of maximum heat capacity. Samples were dialyzed in 10 mM sodium phosphate buffer, pH 7.0, and diluted to 8–10 µM for far-UV circular dichroism analysis. Ellipticity was measured from 250 to 190 nm on an Aviv 202 spectrometer (Aviv Biomedical, Lakewood, NJ) with a quartz cuvette with a 1-mm path length (New Era, Vineland, NJ). Thermal denaturation was conducted by measuring ellipticity at 216 nm from 25 to 95 °C and calculating Tm from a standard two-state unfolding curve.
Thermal stabilities were also determined using a yeast surface display thermal denaturation assay derived from Orr et al
Fn3 was displayed on the yeast surface as for measurement of kinetic and equilibrium binding constants. Cells were washed and resuspended with PBSA, incubated at 20–85 °C for 30 min, and incubated on ice for 5 min. Biotinylated lysozyme was added at a saturating concentration (e.g., 20 nM for L7.5.1) and mouse anti-c-myc
antibody was added at 40 mg/L and incubated on ice for 20 min. Cells were washed and incubated in PBSA with 10 mg/L streptavidin–R-phycoerythrin and 25 mg/L AlexaFluor488 conjugated goat anti-mouse antibody. Cells were washed and resuspended in PBSA and analyzed on an Epics XL flow cytometer. The minimum and maximum fluorescence (Fmin
, respectively), the 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:
L7.5.1 reversion clone construction
Reversion of engineered loops of L7.5.1 to wild-type sequence was accomplished by annealing and extending two PCR products created with a gene-terminal primer and a primer that annealed adjacent to the loop of interest but was extended to include wild-type sequence. Specifically, one PCR reaction contained a 5′ gene terminal primer and a primer that annealed to the 25 nucleotides immediately 5′ of the loop of interest but included a nonannealing ‘tail’ encoding for the wild-type loop sequence. In parallel, PCR was performed with a 3′ gene terminal primer and a primer annealing to the 25 nucleotides immediately 3′ of the loop and including a nonannealing tail. The first PCR product encodes from the start of the gene to the loop of interest and the second PCR product encodes from the loop of interest to the end of the gene. These two products are annealed and extended to yield the full Fn3 gene containing the wild-type sequence in the loop of interest. Framework reversions 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.
Focused library construction
The DE randomization library was created in a manner similar to that of the L7.5.1 loop reversion clones. One PCR amplified the L7.5.1 S15P gene fragment 5′ of the DNA encoding for the DE loop. A second PCR amplified the gene 3′ of the DNA encoding the DE loop using a primer that included a degenerate (NNB) DE loop sequence and 20 nucleotides of overlap with the other PCR product. The PCR products were annealed, extended to produce the full gene, and amplified. This process was conducted independently with four oligonucleotides encoding the four different DE loop lengths. The gene fragments were electroporated into electrocompetent EBY100 along with pCT-Fn3-Loop vector. The resulting library encoded for L7.5.1 S15P with a fully random DE loop of length 4, 5, 6, or 7 amino acids.
A library randomizing the unconserved residues of clones similar to L7.5.1 was constructed by PCR of L7.5.1 S15P The 5′ primer contained 17 nucleotides 5′ of the BC loop, 21 nucleotides to encode the BC loop, and 22 nucleotides to anneal 3′ of the BC loop. The 3′ primer contained 19 nucleotides 3′ of the FG loop, 30 nucleotides to encode for the FG loop, and 10 nucleotides 5′ of the FG loop (note that the nucleotides encoding the first three conserved amino acids of the FG loop also enable annealing during PCR). The PCR products were amplified with extended primers to increase the length of the conserved sequence flanking the loops to improve homologous recombination. The gene fragments were electroporated into electrocompetent EBY100 along with pCT-Fn3-Loop vector. Two versions of the BC and FG loops were included. One oligonucleotide completely randomized the unconserved residues using NNB degeneracy (BC, RXXPWAX; FG, RVGRXXXXXG). The other oligonucleotide restricted diversity to amino acids observed during affinity maturation [BC, R(D/G)(C/H/R/ Y)PWA(I/T); FG, RVG(R/W)(A/M/T/V)(F/L/P/S)(C/D/ G/Y)(A/T)(L/P/S)(G/S)].