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Spy chemistry‐enabled protein directional immobilization and protein purification
Author(s) -
Lin Zhanglin,
Lin Qiao,
Li Jiahui,
Pistolozzi Marco,
Zhao Lei,
Yang Xiaofeng,
Ye Yanrui
Publication year - 2020
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27460
Subject(s) - intein , chemistry , monomer , protein engineering , homogeneous , combinatorial chemistry , biochemistry , chromatography , organic chemistry , polymer , enzyme , thermodynamics , rna , physics , rna splicing , gene
Site‐directed protein immobilization allows the homogeneous orientation of proteins with high retention of activity, which is advantageous for many applications. Here, we report a facile, specific, and efficient strategy based on the SpyTag‐SpyCatcher chemistry. Two SpyTag‐fused model proteins, that is, the monomeric red fluorescent protein (RFP) and the oligomeric glutaryl‐7‐aminocephalosporanic acid acylase, were easily immobilized onto a SpyCatcher‐modified resin directly from cell lysates, with activity recoveries in the range of 85–91%. This strategy was further adapted to protein purification, which proceeded through the selective capture of the SpyCatcher‐fused target proteins by a SpyTag‐modified resin, with the aid of an intein to generate authentic N‐termini. For two model proteins, that is, RFP and a variable domain of a heavy chain antibody, the yields were ∼3–7 mg/L culture with >90% purities. This approach could provide a versatile tool for producing high‐performance immobilized protein devices and proteins for industrial and therapeutic uses.