Page 51 - I. Chemical biology and drug discovery

Development of modified spytag and spycatcher pair for application in affinity

b
a,b
a
chromatography Jin Young Son , Sun Hee Park , Hyo Jin Kang , and Sang J. Chung a*, b*
a College of Pharmacy, Sungkyunkwan University, Suwon, Korea
b AbTis Co. Ltd., Suwon, Korea
BACKGROUND AIM

TheSpyTagandSpyCatchersystemisanefficienttoolforbioconjugationof weaimtomodifythissystemtopreventtheformationoftheirreversible
recombinant peptides. The SpyTag and SpyCatcher protein have relative isopeptide bond. To this end, we designed a mutated Catcher protein
high binding affinity (0.2 µM) and spontaneously form an isopeptide bond. (K31R) which non-covalently binds to the Tag mostly relying on
Our research revolves around the application of this system in protein hydrophobic interactions. In an attempt to improve the binding affinity,
purificationstrategiesemployingaffinitycolumnchromatography. weconstructedthepeptidelibrariesoftheSpyTag.

METHODS

wedesignedamutatedCatcherprotein(K31R)whichnon-covalentlybindstotheTagmostlyrelyingonhydrophobicinteractions.Inan
attempttoimprovethebindingaffinity,weconstructedthepeptidelibrariesoftheSpyTag.BasedontheX-raycrystalstructure,crucial
aminoacidresiduesweresubstitutedforthemorehydrophobicaromaticaminoacids.Atthesametime,toascertainthewatersolubility,
non crucial amino acid residues were replaced by aspartic acid. The peptide libraries were constructed using solid phase peptide
synthesis.Next,thebindingoftheon-beadpeptidelibrarytothefluorescentlabeledCatcherproteinwasscreenedusingafluorescence
microscope.

RESULTS

Table 2. The intensity of 36 peptide library beads
Each of bead was treated with the fluorescently
labeled spy catcher protein (K31R) (Cy 3 NHS ester
was used)(400nM, 2h) and washed. A number of
series show higher intensity than standard peptide
(AHIVMVDAYSG 387).
YWV and WYL give high intensity (YWV 1270, WYL
1135).
Figure1.TheSpyTagandSpyCatchersystemandmutatedSpyCatcher(K31R) FWY, WYY has specially high intensity which can
recognized through fluorescence microscope images
(FWY 1826, WYY 1748).
ㅋ

Figure2..ModifiedstandardSpyTagpeptidewhichislysinesubstitutedtoserineandlibrary
peptidewhichhashydrophobicaromaticaminoacids(36series)

. Figure 5. Fluorescence
HBTU ( 4 0 e q ) .
.
HOBT ( 4 0 e q ) A c 2 O ( 6 0 e q ) . intensity measurement (A)
.
.

-
-
HO PEG 4 OH : moc - PEG 8 OH H2N DIPEA ( 8 0 e q ) DMAP ( 0 1 e q ) 0 2 g N orma l se q uence has lower intensity than library
-
F
. DMF DMF

1 ) 0 5 e q : . e q T en t a g e l res i n 1 ) 13h . peptide beads (C) and (D) has
.

2 ) 0 5 e q : . 0 5 e q 1 7 g 2 ) 8h 1 5 g higher intensity
.

0 5

Lib rar y se q uence
O
O H O H O H O H O H O H O H O H O H O H O O O O O
H 2 N CHC N CHC N CHC N CHC N CHC N CHC N CHC N CHC N CHC N CHC N C C N O O O O N
R CH 2 R CH 2 R CH 2 CH 2 CH 2 CH 2 CH 2 H 2 H H
C O C O C O C O OH
N OH OH OH OH
NH
OH
Figure 3. Reaction Condition The coupling of amino acids was carried out
following standard Fmoc-chemistry with Tentagel resin (loading of 0.26
mmol/g), the protected Fmoc-amino acids (4 eq), HBTU (4 eq), HOBT Figure 8. SPR exprerimet the selected
(4 eq), DIPEA (8 eq), DMF as solvent. Pegylated resin was prepared modifided tag peptide (WYL) has affinity to
with HO-PEG4, Fmoc-NH 2 -PEG 8 . and deprotected with 20% piperidine mutated spy catcher protein (K31R). SPR
in DMF. One cycle of peptide elongation was carried out the mentioned exprerimet was planned to immobilize tag
protocol (0.2 M in DMF). Resin was washed with DMF(1 time, 10min peptide and flow catcher protein. The
and 3 times, 10 sec) and DCM(3 times, 10 sec) in each of elongation exprerimet conducted with
and deprotection step. Instrument :Reichert SR7500DC system, Software:
Scrubber2, Sensor Chip: CMDH chip
CONCLUSION REFERENCES ACKNOWLEDGEMENTS
Using Fluorescently labeled proteins, we can 1. Zakeri, Bijan, et al. "Peptide tag forming a rapid covalent This work was supported by National Research Foundation
identify affinity differences with less disturbance of bond to a protein, through engineering a bacterial adhesin." of Korea (NRF) grants (NRF-2017M3A9C8031995) and the
free dye. Proceedings of the National Academy of Sciences 109.12 Bio & Medical Technology Development Program of the
SPR result proved that selected modified tag (2012): E690-E697. National Research Foundation (NRF) funded by the Korean
peptide (WYL) has affinity (KD = 1.45(2) uM) to government (MSIT) (NRF-2012M3A9C4048775)
mutated spy catcher protein (K31R).
We are planning to check affinity on other peptides Contact information
and standard peptide with SPR experiment and
show higher intensity difference with RFP fusion
proteins. E-mail: sjchung@skku.edu
.