Page 52 - I. Chemical biology and drug discovery

[I. Chemical biology and drug discovery-29]

Development of modified spytag and spycatcher pair for

application in affinity chromatography

Jin Young Son¹, Sun Hee Park¹˙², Hyo Jin Kang², Sang Jeon Chung¹˙²˙*

¹Pharmacy, Sungkyunkwan University, Suwon 16419, South Korea, ²AbTis Co. Ltd, AbTis Co. Ltd, Suwon 16648,

South Korea

The SpyTag and SpyCatcher system is an efficient tool for bioconjugation of recombinant peptides. The SpyTag and
SpyCatcher protein have relative high binding affinity (0.2 µM) and spontaneously form an isopeptide bond.1 Our

research revolves around the application of this system in protein purification strategies employing affinity column

chromatography. For this purpose, we aim to modify this system to prevent the formation of the irreversible
isopeptide bond. To this end, we designed a mutated Catcher protein (K10R) which non-covalently binds to the Tag
mostly relying on hydrophobic interactions. In an attempt to improve the binding affinity, we constructed the

peptide libraries of the SpyTag. Based on the X-ray crystal structure, crucial amino acid residues were substituted
for the more hydrophobic aromatic amino acids . At the same time, to ascertain the water solubility, non crucial

amino acid residues were replaced by aspartic acid. The peptide libraries were constructed using solid phase peptide
synthesis. Next, the binding of the on-bead peptide library to the fluorescent labeled Catcher protein was screened

using a fluorescence microscope. In conclusion, Several modified Tag peptides were found to have improved binding
with respect to the non-modified Tag peptide.