Improved variants of SrtA for site-specific conjugation on antibodies and proteins with high efficiency

被引：86

作者：

Chen, Long ^{[1
]}

Cohen, Justin ^{[2
]}

Song, Xiaoda ^{[3
]}

Zhao, Aishan ^{[1
]}

Ye, Zi ^{[1
]}

Feulner, Christine J. ^{[2
]}

Doonan, Patrick ^{[2
]}

Somers, Will ^{[2
]}

Lin, Laura ^{[2
]}

Chen, Peng R. ^{[1
,4
]}

机构：

[1] Peking Univ, Beijing Natl Lab Mol Sci, Coll Chem & Mol Engn, Synthet & Funct Biomol Ctr, Beijing 100871, Peoples R China

[2] Pfizer Inc, Dept Global Biotherapeut Technol, Cambridge, MA 02140 USA

[3] Nanjing Univ, Sch Life Sci, Nanjing, Jiangsu, Peoples R China

[4] Peking Tsinghua Ctr Life Sci, Beijing, Peoples R China

来源：

SCIENTIFIC REPORTS | 2016年 / 6卷

关键词：

MONOCLONAL-ANTIBODY; CYTOTOXIC DRUG; SORTASE; IDENTIFICATION; RESIDUES; CANCER;

D O I：

10.1038/srep31899

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

Sortase mediated ligation is a highly specific platform for conjugation that relies on the specificity of the transpeptidase Sortase A ( SrtA) for short peptide sequences ( LPXTG and GGG). SrtA retains its specificity while accepting a wide range of potential substrates, but its broad use is limited by the wild-type enzyme's poor kinetics, which require large amounts of SrtA and extended reaction times for efficient conjugation. Prior explorations have aimed to improve the kinetics of SrtA with limited success. Herein we describe the discovery of further improved SrtA variants with increased efficiency for the conjugation reaction, and demonstrate their robustness in labelling proteins and antibodies in a site-specific manner. Our variants require significantly lower amounts of enzyme than WT SrtA and can be used to attach small molecules to the N or C-terminus of the heavy or light chain in antibodies with excellent yields. These improved variants can also be used for highly efficient site-specific PEGylation.

引用

页数：12

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