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Supplemental Data Methods and Materials:
Materials and Methods
pYD41 yeast display vector (kind gift from Drs. Susan Lacy and Jon Belk, Abbott) is
derived from pYD1 vector (Invitrogen) and introduces Sfi1, Nco1, Xho1, and Not1
restriction sites into polylinker.
EBY100 (Trp- phenotype) yeast strain (supplied with pYD1 vector from Invitrogen).
Strain genotype: MATa ura3-52 trp1 leu2 1 his3 200 pep4::HIS3 prb11.6R can1
GAL (pIU211:URA3).
Mutagenic library construction
Linearized pYD41 vector missing specific regions of each CDR to be replaced (“gapped”
vector) were prepared by “reverse” PCR. Tacrolimus WT scFv pYD41 plasmid DNA (1
ng/rx) was used as template and amplified with 0.2 mM CDR for gap primer (anneals on
sense strand at 3’ end of CDR to be replaced) and 0.2 mM CDR rev gap primer (anneals
on anti-sense strand at 5’ end of CDR to be replaced) using 5 units of LA Taq polymerase
(Takara Bio). Gapped vector DNA was amplified for 10 cycles of 30 sec at 94oC, 30 sec
at 60oC, and 6 min at 68oC followed by 20 cycles of 30 sec at 94oC, 30 sec at 60oC, and 6
min + 20 sec per additional cycle at 68oC. The amplification products (H1 gap, H2 gap,
H3 gap, L1 gap, L2 gap, and L3 gap vectors) were gel purified and 1 μg of linearized
“gapped” vector was co-transformed with 16 μg of the appropriate degenerate ssDNA
oligonucleotide (see Supplemental Table 1) to create each of the 51 mutagenic libraries
as outlined in Methods section.
Generation of tacrolimus combinatorial mutant clones
Portions of yeast colony from each individual clone were boiled for 5 min in 20 μl of
0.1% SDS and 2 μl of the resulting lysate used as template after centrifugation. Initially,
each clone isolated after selection was amplified with 0.25 mM appropriate for and rev
primer, 0.2 mM dNTPs, 2 mM MgSO4, 0.1% TX-100 and 1 unit HiFi Taq polymerase in
1x HiFi buffer for 30 cycles of 15 sec at 94oC, 30 sec at 55oC, and 1 min at 68oC.
Sequences of amplification primers are shown in supplemental table 2 and the various
initial mutant PCR products listed in supplemental table 3. In order to create VL genes
combining the L1 mutation with the L3 cys or ser mutations, representative portions of
each VL mutant were amplified with homology in the framework 3 region and then
combined by SOE-PCR using VL gap for and rev primers using conditions outlined
above to reconstruct intact VL genes. Intact scFv containing the various mutant
combinations (supplemental table 3) were reconstructed by SOE-PCR via homology
between the VH and VL genes in the linker region using VH gap for and VL gap rev
primers using conditions outlined above.
Binding analysis of scFv clones
A first order exponential decay equation was used to fit the antigen-binding mean
fluorescence intensity (MFI) obtained from each time point:
F  Fsat
 k
o ff
t
F
bkg
(1)
where, F is observed fluorescence, Fsat is fluorescence at saturation prior to competitor
addition, Fbkg is background fluorescence of cells, koff is dissociation rate constant and t is
time in seconds.
The dissociation half-life (t1/2) was determined using the derived dissociation rate using
equation 2:
t1 / 2  ln 2 / koff
(2).
The MFI of the antigen-binding channel was fit to a nonlinear least-squares regression to
calculate the equilibrium affinity constant (KD)
F  Fbkg 
Fsat  [ L]
K D  [ L]
where, F is observed fluorescence, Fsat is fluorescence at maximal binding, Fbkg is
background fluorescence of cells, [L] is concentration of antigen, and KD is the
equilibrium dissociation constant.
(3)
Supplemental Table 1. Mutagenic oligonucleotides. Gap oligos flank the region to be
mutagenized and are used for “reverse” PCR amplification to create a “gapped” vector.
The CDR regions are underlined and mutagenic window indicated with NNS degenerate
codon for each mutagenic oligo. Regions of homology used for recombination to insert
the mutant oligo into “gapped” scFv gene flank the CDR region. All sequences are listed
5’ to 3’.
Oligo Name
Sequence
H1 gap for
TGGGTTCGCCAGACGCC
H1 gap rev
AGAGGCTGCACAGGAGAGTTTCA
H1-1
H1-2
H1-3
H1-4
H1-5
H1-6
H1-7
H1-8
H2 gap for
H2 gap rev
H2-1
H2-2
H2-3
H2-4
H2-5
H2-6
H2-7
H2-8
H3 gap for
H3 gap rev
H3-1
H3-2
H3-3
H3-4
H3-5
H3-6
H3-7
H3-8
CTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTNNSNNSNNSTTCAGTAGTTATGGCATGTCTTGGGTTCGCC
AGACGCCAGACAAGAGGCTGGAGTG
CTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGANNSNNSNNSAGTAGTTATGGCATGTCTTGGGTTCGCC
AGACGCCAGACAAGAGGCTGGAGTG
CTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCNNSNNSNNSAGTTATGGCATGTCTTGGGTTCGCC
AGACGCCAGACAAGAGGCTGGAGTG
CTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTNNSNNSNNSTATGGCATGTCTTGGGTTCGCC
AGACGCCAGACAAGAGGCTGGAGTG
CTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCNNSNNSNNSGGCATGTCTTGGGTTCGCC
AGACGCCAGACAAGAGGCTGGAGTG
CTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTNNSNNSNNSATGTCTTGGGTTCGCC
AGACGCCAGACAAGAGGCTGGAGTG
CTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGTNNSNNSNNSTCTTGGGTTCGCC
AGACGCCAGACAAGAGGCTGGAGTG
CTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGTTATNNSNNSNNSTGGGTTCGCC
AGACGCCAGACAAGAGGCTGGAGTG
TATCCAGACAGTGTGAAGGGGC
TGCGACCCACTCCAGCCT
GCCAGACGCCAGACAAGAGGCTGGAGTGGGTCGCANNSNNSNNSAGTGGTGGTACTTACACCTTCTATCCAGACA
GTGTGAAGGGGCGCTTCACCATCTC
GCCAGACGCCAGACAAGAGGCTGGAGTGGGTCGCAACCNNSNNSNNSGGTGGTACTTACACCTTCTATCCAGACA
GTGTGAAGGGGCGCTTCACCATCTC
GCCAGACGCCAGACAAGAGGCTGGAGTGGGTCGCAACCATTNNSNNSNNSGGTACTTACACCTTCTATCCAGACA
GTGTGAAGGGGCGCTTCACCATCTC
GCCAGACGCCAGACAAGAGGCTGGAGTGGGTCGCAACCATTAGTNNSNNSNNSACTTACACCTTCTATCCAGACA
GTGTGAAGGGGCGCTTCACCATCTC
GCCAGACGCCAGACAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTNNSNNSNNSTACACCTTCTATCCAGACA
GTGTGAAGGGGCGCTTCACCATCTC
GCCAGACGCCAGACAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTNNSNNSNNSACCTTCTATCCAGACA
GTGTGAAGGGGCGCTTCACCATCTC
GCCAGACGCCAGACAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTNNSNNSNNSTTCTATCCAGACA
GTGTGAAGGGGCGCTTCACCATCTC
GCCAGACGCCAGACAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTACTNNSNNSNNSTATCCAGACA
GTGTGAAGGGGCGCTTCACCATCTC
TGGGGCCAAGGGACTCTG
TCTTGAACAGTAATACATGGCTGTGT
AGTCTGCAGACACAGCCATGTATTACTGTTCAAGANNSNNSNNSGGTTACTCCTGGTTTCCTTATTGGGGCCAAG
GGACTCTGGTCACTGTCTCTGCAGG
AGTCTGCAGACACAGCCATGTATTACTGTTCAAGACAGNNSNNSNNSTACTCCTGGTTTCCTTATTGGGGCCAAG
GGACTCTGGTCACTGTCTCTGCAGG
AGTCTGCAGACACAGCCATGTATTACTGTTCAAGACAGACCNNSNNSNNSTCCTGGTTTCCTTATTGGGGCCAAG
GGACTCTGGTCACTGTCTCTGCAGG
AGTCTGCAGACACAGCCATGTATTACTGTTCAAGACAGACCGATNNSNNSNNSTGGTTTCCTTATTGGGGCCAAG
GGACTCTGGTCACTGTCTCTGCAGG
AGTCTGCAGACACAGCCATGTATTACTGTTCAAGACAGACCGATGGTNNSNNSNNSTTTCCTTATTGGGGCCAAG
GGACTCTGGTCACTGTCTCTGCAGG
AGTCTGCAGACACAGCCATGTATTACTGTTCAAGACAGACCGATGGTTACNNSNNSNNSCCTTATTGGGGCCAAG
GGACTCTGGTCACTGTCTCTGCAGG
AGTCTGCAGACACAGCCATGTATTACTGTTCAAGACAGACCGATGGTTACTCCNNSNNSNNSTATTGGGGCCAAG
GGACTCTGGTCACTGTCTCTGCAGG
AGTCTGCAGACACAGCCATGTATTACTGTTCAAGACAGACCGATGGTTACTCCTGGNNSNNSNNSTGGGGCCAAG
GGACTCTGGTCACTGTCTCTGCAGG
L1 gap for-1 AGTACTGGAAACACCTTTTTAGAATGG
L1 gap rev-1 GCAAGAGATGGAGGCTTGATC
L1-1
L1-2
L1-3
L1-4
L1-5
L1-6
TGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCNNSNNSNNSCAGAGCATTGTACATAGTACTGGAAACA
CCTTTTTAGAATGGTTTTTGCAGAA
TGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAAANNSNNSNNSAGCATTGTACATAGTACTGGAAACA
CCTTTTTAGAATGGTTTTTGCAGAA
TGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAAATCTNNSNNSNNSATTGTACATAGTACTGGAAACA
CCTTTTTAGAATGGTTTTTGCAGAA
TGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAAATCTAGTNNSNNSNNSGTACATAGTACTGGAAACA
CCTTTTTAGAATGGTTTTTGCAGAA
TGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAAATCTAGTCAGNNSNNSNNSCATAGTACTGGAAACA
CCTTTTTAGAATGGTTTTTGCAGAA
TGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAAATCTAGTCAGAGCNNSNNSNNSAGTACTGGAAACA
CCTTTTTAGAATGGTTTTTGCAGAA
TGGTTTTTGCAGAAGCCAGG
L1 gap for-2
L1 gap rev-2 AATGCTCTGACTAGATTTGCAAGAGA
L1-7
L1-8
L1-9
L1-10
L1-11
L1-12
L1-13
L1-14
L2 gap for
L2 gap rev
L2-1
L2-2
L2-3
L2-4
L2-5
L3 gap for
L3 gap rev
L3-1
L3-2
L3-3
L3-4
L3-5
L3-6
L3-7
AAGCCTCCATCTCTTGCAAATCTAGTCAGAGCATTNNSNNSNNSACTGGAAACACCTTTTTAGAATGGTTTTTGC
AGAAGCCAGGCCAGTCTCCAAAGCT
AAGCCTCCATCTCTTGCAAATCTAGTCAGAGCATTGTANNSNNSNNSGGAAACACCTTTTTAGAATGGTTTTTGC
AGAAGCCAGGCCAGTCTCCAAAGCT
AAGCCTCCATCTCTTGCAAATCTAGTCAGAGCATTGTACATNNSNNSNNSAACACCTTTTTAGAATGGTTTTTGC
AGAAGCCAGGCCAGTCTCCAAAGCT
AAGCCTCCATCTCTTGCAAATCTAGTCAGAGCATTGTACATAGTNNSNNSNNSACCTTTTTAGAATGGTTTTTGC
AGAAGCCAGGCCAGTCTCCAAAGCT
AAGCCTCCATCTCTTGCAAATCTAGTCAGAGCATTGTACATAGTACTNNSNNSNNSTTTTTAGAATGGTTTTTGC
AGAAGCCAGGCCAGTCTCCAAAGCT
AAGCCTCCATCTCTTGCAAATCTAGTCAGAGCATTGTACATAGTACTGGANNSNNSNNSTTAGAATGGTTTTTGC
AGAAGCCAGGCCAGTCTCCAAAGCT
AAGCCTCCATCTCTTGCAAATCTAGTCAGAGCATTGTACATAGTACTGGAAACNNSNNSNNSGAATGGTTTTTGC
AGAAGCCAGGCCAGTCTCCAAAGCT
AAGCCTCCATCTCTTGCAAATCTAGTCAGAGCATTGTACATAGTACTGGAAACACCNNSNNSNNSTGGTTTTTGC
AGAAGCCAGGCCAGTCTCCAAAGCT
GGGGTCCCAGACAGGTTCAGT
GTAGATCAGGAGCTTTGGAGACTGG
TTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACNNSNNSNNSAACCGATTTTCTGGGGTCCCAGACAGG
TTCAGTGGCAGTGGATCAGGGACAG
TTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAANNSNNSNNSCGATTTTCTGGGGTCCCAGACAGG
TTCAGTGGCAGTGGATCAGGGACAG
TTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAATTNNSNNSNNSTTTTCTGGGGTCCCAGACAGG
TTCAGTGGCAGTGGATCAGGGACAG
TTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAATTTCCNNSNNSNNSTCTGGGGTCCCAGACAGG
TTCAGTGGCAGTGGATCAGGGACAG
TTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAATTTCCAACNNSNNSNNSGGGGTCCCAGACAGG
TTCAGTGGCAGTGGATCAGGGACAG
TTCGGTGCTGGGACCAAGC
GCAGTAATAAACTCCCAGATCCTCAGAC
AGAGTGGAGTCTGAGGATCTGGGAGTTTATTACTGCNNSNNSNNSTCACATGTTCCGCTCACGTTCGGTGCTGGG
ACCAAGCTGGAGCTGAAACGGGCG
AGAGTGGAGTCTGAGGATCTGGGAGTTTATTACTGCTTTNNSNNSNNSCATGTTCCGCTCACGTTCGGTGCTGGG
ACCAAGCTGGAGCTGAAACGGGCG
AGAGTGGAGTCTGAGGATCTGGGAGTTTATTACTGCTTTCAANNSNNSNNSGTTCCGCTCACGTTCGGTGCTGGG
ACCAAGCTGGAGCTGAAACGGGCG
AGAGTGGAGTCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTNNSNNSNNSCCGCTCACGTTCGGTGCTGGG
ACCAAGCTGGAGCTGAAACGGGCG
AGAGTGGAGTCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCANNSNNSNNSCTCACGTTCGGTGCTGGG
ACCAAGCTGGAGCTGAAACGGGCG
AGAGTGGAGTCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATNNSNNSNNSACGTTCGGTGCTGGG
ACCAAGCTGGAGCTGAAACGGGCG
AGAGTGGAGTCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTNNSNNSNNSTTCGGTGCTGGG
ACCAAGCTGGAGCTGAAACGGGCG
Supplemental Table 2. Primers for combinatorial mutant SOE-PCR
Oligo Name
VH gap for
VH gap rev
VL gap for
VL gap rev
Tacro L1 gap rev
Tacro L3 gap for
Sequence
ATGACTGGTGGACAGCAAATGGG
AGAGACCTTCGCCTCCTTCAGGG
GGTCCCGCCAAGGAGTTGAC
CGTAGAATCGAGACCGAGGAGA
CACTGAACCTGTCTGGGACCCC
CCAGGCCAGTCTCCAAAGCTC
Supplemental Table 3. Combinatorial mutant SOE-PCR products. Individual mutant
variable domains, or fragments thereof, are listed in top portion of table. The
reconstructed intact combinatorial mutant scFv genes are listed in bottom portion of
table. The template(s) and primers used for PCR, as well as, resulting product are listed
in each column, respectively.
Template
For primer
Rev primer
Product
H2-1A
L1-1B
L1-1B
L3-1A
L3-2B
L1-1B
L3-1A
L3-2B
L1 frag
L3 cys frag
L1 frag
L3 ser frag
H2 VH
L3 cys VL
H2 VH
L3 ser VL
WT VH
L1-L3 cys VL
WT VH
L1-L3 ser VL
H2 VH
L1-L3 ala VL
H2 VH
L1-L3 cys VL
H2 VH
L1-L3 ser VL
VH gap for
VH gap for
VL gap for
VL gap for
VL gap for
VL gap for
Tacro L3 gap for
Tacro L3 gap for
VH gap rev
VH gap rev
VL gap rev
VL gap rev
VL gap rev
Tacro L1 gap rev
VL gap rev
VL gap rev
H2 VH
WT VH
L1-L3 ala VL
L3 cys VL
L3 ser VL
L1 fragment
L3 cys fragment
L3 ser fragment
VL gap for
VL gap rev
L1-L3 cys VL
VL gap for
VL gap rev
L1-L3 ser VL
VH gap for
VL gap rev
H2-L3 cys scFv
VH gap for
VL gap rev
H2-L3 ser scFv
VH gap for
VL gap rev
L1-L3 cys scFv
VH gap for
VL gap rev
L1-L3 ser scFv
VH gap for
VL gap rev
H2-L1-L3 ala scFv
VH gap for
VL gap rev
H2-L1-L3 cys scFv
VH gap for
VL gap rev
H2-L1-L3 ser scFv