| Autor: |
Lin, Zhanglin, Lin, Qiao, Li, Jiahui, Pistolozzi, Marco, Zhao, Lei, Yang, Xiaofeng, Ye, Yanrui |
| Zdroj: |
Biotechnology & Bioengineering; Oct2020, Vol. 117 Issue 10, p2923-2932, 10p |
| Abstrakt: |
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. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
|
Plný text