Strains, media and antibodies
The yeast strain EBY100Zeo was a gift from Dr. Feldhaus (Pacific Northwest National laboratory, Richland, WA). Briefly, EBY100Zeo was derived from EBY100 (GAL1-AGA1:URA3 ura3-52 trp1 leu2Δ1 his3Δ200 pep4::HIS2 prb1Δ1.6R can1 GAL
) and carries the pTEF1 promoter Zeocin resistance gene (Sh ble
gene). EBY100Zeo was maintained in YPD media (Current Protocols in Molecular Biology
, John Wiley and Sons, Chapter 13.1.2). The bacterial strain E. coli
E, thi, hsd
ZΔM15) was used for cloning and preparation of plasmid DNA. The gene fragment encoding for botulinum neurotoxin type A (Hall) (BoNT/A) binding domain (HC
) and the expression plasmid pET24a/(LC
) harboring the gene fragment that encodes for the fusion of light chain (LC
) and translocation domains (HN
) of BoNT/A were kindly provided by Dr. Leonard Smith (USAMRIID, MD)44-48
. LiAc–treated EBY100Zeo cells were transformed, as previously described 49
, with plasmid derivatives of the pYD2 yeast display vector (see below) and selected on SD-CAA medium (Current Protocols in Molecular Biology
, John Wiley and Sons, Chapter 13.1.2). HC
yeast surface display was induced by transferring yeast cultures from SD-CAA to SG-CAA medium (identical to SD-CAA medium except the glucose was replaced by galactose) supplemented with 12.5 μg ml−1
tetracycline, 50 μg ml−1
kanamycin and 25 μg ml−1
zeocin and growing at 18°C for 48 h, as described 50
BoNT/A neutralizing monoclonal antibodies (mAbs) 3D12, HuC25, AR2, ING2, ING1, 9D8, and S25 were used 17, 22, 25
. For IgG and FAB
fragment detection by flow cytometry (FACS), Phycoerythrin (PE)-labeled goat-anti-human IgG, PE-labeled goat-anti-mouse IgG or FAB
-specific Allophycocyanin (APC-647nm)-conjugated goat anti-human F(AB′)2
were employed (Jackson ImmunoResearch Laboratories, PA). Expression in yeast was monitored using an SV5 antibody that was purified from hybridoma supernatant with a Protein G column (GE Healthcare, NJ) and then directly labeled with Alexa-488 or Alexa-647 with a kit provided by Molecular Probes (Carlsbad, CA).
The following oligonucleotides were purchased from Sigma-Proligo (St. Louis, MO):
LCHnFor: 5′-ATATATAATCCATGGTTCAGTTCG TTAACAAACAGTTCAACTAC-3′
Construction and expression of plasmids for yeast display of BoNT/A domains
The yeast display vector pYD2 was derived from pYD1 (Invitrogen, Carlsbad, CA), as previously described 22
. Primers HCFor and HCRev were designed to PCR amplify the HC
gene fragment, adding the restriction sites NcoI
. The resulting PCR product and pYD2 were then both digested with NcoI
and their ligation resulted in the yeast display vector pYD2/HC
. Primers LCHnFor and LCRev were designed to PCR amplify the LC
gene fragment from the expression vector pET24a/(LC
), adding the restriction sites NcoI
. Following digestion of both pYD2 and the resulting PCR amplification product with NcoI
was gel-purified and ligated into pYD2 to yield the yeast display vector pYD2/LC
. Similarly, reverse primer LCHnRev and forward primers HNBeltFor or HCFor were used in order to PCR amplify the gene fragments encoding for the translocation domain HN
with or without the belt region, respectively. Subsequently, ligation into pYD2 yielded the yeast display vectors pYD2/HN
(including the belt) and pYD2/HN
(without the belt). In order to construct the plasmid pYD2/LC
, primers LCFor and LCHnRev were first used to PCR amplify a C-terminal portion of LC
from plasmid pET24a/LC
adding the restriction sites BsrGI
Both the PCR amplification product and pYD2/LC
were digested with BsrGI
purified and then ligated together to yield the final vector pYD2/LC
For domain epitope mapping of BoNT/A antibodies, the plasmids pYD2/HC, pYD2/LC, pYD2/HN (including the belt), pYD2/HN (without the belt) and pYD2/LC-HN were transformed to LiAc-treated EBY100Zeo cells. Yeast cultures were then grown and induced, as described above. To quantitate binding to botulinum neurotoxin domains, ING2 that recognizes BoNT/A LC, ING1 and 9D8 recognizing BoNT/A HN, HuC25, AR2 and 3D12 recognizing BoNT/A1 HC were used. Purified 3D12 and AR2 were directly labeled with Alexa-647 (Molecular Probes, CA). Purified HuC25, ING1, and ING2 were indirectly labeled with PE-goat-anti-human IgG and 9D8 with PE-goat-anti-mouse IgG.
To measure antibody binding within the yeast surface display context, only domain displaying yeast (binding to SV5 mAb) were included in the analysis by co-staining with SV5 (Alexa-488) or SV5 (Alexa-647).
Construction and expression of a mutant BoNT/A HC yeast displayed library
A library of HC
mutants was generated by random mutagenesis using error prone PCR, as described by Fromant and coworkers 26
. Briefly, primers Gap5 and Gap3 and Taq polymerase were used to PCR amplify HC
from the plasmid pYD2/HC
in a reaction mixture which included 0.5 mM MnCl2
and an excess concentration of one of the four substrate dNTPs. The amplified mutated HC
PCR product was then gel purified using a gel extraction kit by Qiagen (Valencia, CA) and approximately 12 μg of HC
and 12 μg of NcoI-NotI
-digested-pYD2 vector (3:1 insert:vector molar ratio) were used to transform LiAc-treated EBY100Zeo cells by gap repair. Transformation mixtures were cultured and subcultured in SD-CAA media. The size of the library in yeast was calculated from the number of colonies appearing on SD-CAA plates that were plated with serial dilutions of the transformation mixture. The estimated error-rate of the mutagenic library was determined by colony-PCR amplification from single colonies with primers PYDFor and GAP3Rev, followed by sequencing (Elim Biopharmaceuticals, CA) with primer PYDFor and GAP31Rev. When the OD600
of the subcultured library reached 1, HC
yeast surface display was induced by culturing in SG-CAA media for 48 hours at 18°C.
Selection and analysis of mutant HC libraries by FACS
In order to maintain the library or sort output diversity, an amount of yeast at least ten times larger than the library size or the sort output from the previous round were washed and resuspended in FACS buffer (phosphate-buffered saline (pH 7.4), 0.5% bovine serum albumin).
For the first round of selection, yeast were stained with AR2 (Alexa-647), 3D12 (Alexa-647) and SV5 (Alexa-488) at 1:200 dilution (in FACS buffer) for 2 h at 4°C. All yeast displaying HC binding AR2 and/or 3D12, regardless of display level (SV5 staining) were gated for selection on a FACSAria sorter (BD Biosciences, MD). For the second round of sorting, yeast were stained with 3D12 (Alexa-647) and AR2 (Alexa-488) and sort gates were set to collect yeast displaying HC binding 3D12 or AR2. For the final round of sorting, induced yeast cells were incubated with AR2 (Alexa-488), 3D12 (Alexa-647) and unlabeled SV5 at 1:200 dilution (in FACS buffer) for 2 h at 4°C. Cells were then washed and incubated with the secondary antibody PE-goat-anti-mouse IgG at 1:300 dilution for 1 h at 4°C. Yeast cells were then washed and resuspended in ice-cold FACS buffer. Gates on the FACSAria were set so that only yeast displaying HC that bound exclusively to either AR2 (Alexa-488) or to 3D12 (Alexa-647) would be selected (see ). Fluorescence compensation on FACSAria was necessary in order to subtract the inherent overlap of emission spectra that originate in the antibody PE and Alexa-488 fluorescent labels. Independent gates were set to collect separately yeast displaying HC that were poorly displayed or had no detectable display as measured by SV5 binding.
Following the third round of selection, individual clones from the sort output were inoculated in SD-CAA and then induced in SG-CAA media, as previously described. Induced cells were washed, resuspended in FACS buffer and incubated for 2 h at room temperature with 500 pM HuC25 IgG or 500 pM 3D12 IgG, followed by 1 h incubation with SV5 (Alexa-488) IgG and APC-goat anti-human FAB at 1:200 dilution at 4°C. Plasmids from yeast clones binding exclusively to HuC25 or 3D12 antibodies were recovered using a modified protocol of Qiagen (Valencia, CA) which uses acid-washed glass beads (Sigma, MO) for the efficient lysis of yeast cells. Recovered plasmids were then transformed into chemically competent E. coli DH5α cells. A high copy number of these plasmids could then be recovered using a miniprep kit (Qiagen, CA) and their sequences were confirmed by DNA sequencing (Elim Biopharmaceuticals, CA) with primers PYDFor and PYDRev.
Construction and yeast display of BoNT/A HC single alanine mutants
Genes encoding botulinum neurotoxin HC fragments with a single alanine substitution were constructed using a one-step polymerase chain reaction (PCR). Complementary sets of primers (HCAlaFor and HCAlaRev) were designed so that they could generate a single alanine substitution at the desired amino acid position of the HC gene fragment.
First, PYDFor and HCAlaRev (a different primer for each alanine mutation) were used to PCR amplify the 5′ end part of the HC gene from pYD2/HC, introducing an alanine mutation into the desired amino acid location. Similarly, PYDRev and HCAlaFor (a different primer for each alanine mutation) were used to PCR amplify the 3′ end portion of the HC gene (). The 5′and 3′ end PCR products were then gel-purified using a gel extraction kit (Qiagen, CA) and were used to co-transform LiAc-treated EBY100Zeo cells with NcoI-NotI-digested pYD2 vector. The pYD2-HC (with alanine substitution) plasmids resulting from gap repair were then recovered from yeast cells, re-transformed into DH5α E. coli cells and their sequences were confirmed by DNA sequencing (Elim Biopharmaceuticals, CA) using PYDFor and PYDRev primers, as previously described.
Construction of BoNT/A1 HC mutants with BoNT/A2 amino acid substitutions
Similarly to the construction of the BoNT/A1 alanine mutants, four BoNT/A1 HC mutants were generated with BoNT/A2 substitutions at the specified amino acid locations: a) Y1117F, b) Q1254L, F1255Y, N1256D, c) I1271V, E1272G, R1273K, S1274A, d) R1294S, P1295S. The four pYD2-HC plasmids (with the BoNT/A2 substitutions) resulting from gap-repair were recovered from yeast cells and their sequences were confirmed with PYDFor and PYDRev primers, as previously described.
Generation and purification of FAB from IgG
FAB fragments were prepared from purified IgG using immobilized papain (Pierce Biotechnology, IL). Briefly, IgG was concentrated to ~12 mg ml−1 in 20 mM phosphate, 10 mM EDTA pH 7.0, then added to an equal volume of immobilized papain resin (washed with 20mM phosphate, 10 mM EDTA, 20 mM cysteine pH 7.0) and incubated at 37°C for 16 hours. The immobilized papain was removed by centrifugation, and the digest supernatant was dialyzed against 10 mM MES pH 5.6. The FAB fragment was separated from undigested IgG and FC fragments by cation exchange chromatography (HiTrap SP HP, GE Healthcare, NJ) using a salt gradient. The purified FAB was then dialyzed against PBS and stored at −80°C.
To ensure that the FAB
retained the expected affinity, the KD
of HuC25 and 3D12 IgG and FAB
fragments for yeast-displayed HC
were measured by flow cytometry. The measured KD
values for 3D12 and HuC25 IgGs (90 pM and 20 pM respectively) were comparable to the solution KD
previously measured (61 and 45 pM respectively) 22
. As expected, the equilibrium KD
of the monovalent 3D12 and HuC25 FAB
fragments on yeast displayed HC
, 175pM and 107 pM respectively, were also comparable to the solution KD
but lower than the KD
values of their bivalent IgG counterparts.
Affinity (KD) measurement of FAB fragments on wild type HC and alanine mutants
The dissociation equilibrium constants (KD) of 3D12 and HuC25 FAB fragments for the wild type or alanine-substituted mutants of yeast displayed HC were measured by flow cytometry on a LSRII flow cytometer (BD Biosciences, MD). First, EBY100Zeo yeast cultures harboring the pYD2/HC (wild type) or the pYD2/HC (alanine substituted) plasmids were grown and then induced as described above. Aliquots of approximately 1 × 105 induced yeast cells (i.e., ~0.005 OD600 ml−1) were washed in FACS buffer and incubated with dilutions (ranging from 1μM-16pM) of 3D12 or HuC25 FAB fragments such that the KD would be spanned by at least 10-fold, where possible. Incubation volumes were chosen to ensure that a 10-fold molar excess of the antibody (ligand) over the displayed moiety (HC) would be maintained. For this purpose, we assumed that ~105 HC copies are displayed on the surface of a yeast cell. Incubation with 3D12 or HuC25 FAB was allowed to proceed for 2 hours at room temperature. At that point, cells were washed in FACS buffer and then resuspended in secondary APC-conjugated FAB-specific goat-anti-human F(AB)′2 at 1:200 dilution in FACS buffer. To measure the KD of 3D12 and HuC25 FAB fragments within the surface display context, only the HC displaying yeast were included in the analysis by co-staining with SV5 (Alexa-488) mAb.
Changes in free energy of binding (ΔΔGmut-wt)
For each HC alanine mutation that significantly decreased 3D12 or HuC25 FAB binding, the change of free energy (ΔΔGmut-wt) between the HC alanine (Ala) mutant relative to that of the wild type (wt) was calculated using the following standard formula and substituting with the previously measured KD constants:
These ΔΔGmut-wt calculations provided us with a measure of the energetic contribution of each one of the alanine-substituted amino acid residues on 3D12 or HuC25 binding, therefore indicating the position of their functional epitopes.
Competition of 3D12 FAB with a synthetic peptide epitope
The synthetic peptide N-KYVDVNNVGIRGYMYLKGP-C was purchased from Genemed Synthesis (South San Francisco, CA). Serial dilutions (ranging from 1 to 300μM) of the peptide in PBS buffer solution were allowed to interact for 1h at room temperature with KD concentration of 3D12 FAB. Following incubation with the 3D12 FAB, the 3D12 FAB-peptide mixtures were added to 106 HC-displaying EBY100Zeo (pYD2-HC) yeast cells for 45 min at room temperature. Cells were then washed, incubated for 30 min with secondary APC-conjugated FAB-specific goat-anti-human F(AB)′2 at 1:200 dilution in FACS buffer and SV5 (Alexa-488) and analyzed by FACS.