Intermolecular disulfide bonds between unpaired cysteines retard the C-terminal trans-cleavage of Npu DnaE

Author:

Xu, Yanran

Zhang, Lei

Ma, Buyong

Hu, Lifu

Lu, Huili

Dou, Tonglu

Chen, Junsheng

Zhu, Jianwei
Author Address

Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China., Basic Science Program, Leidos Biomedical Research, Inc. Cancer and Inflammation Program, National Cancer Institute, Frederick, MD, 21702, USA., Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China. Electronic address: js.chan@sjtu.edu.cn., Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China; Jecho Laboratories, Inc., Frederick, MD, USA. Electronic address: jianweiz@sjtu.edu.cn.,

Abstract:

Npu DnaE is a naturally occurred split intein possessing robust trans-splicing activity and could be engineered to perform rapid C-terminal cleavage module by a single mutation D118G. Unfortunately, however, for this modified selfcleaving module, reducing agents were needed to trigger the rapid cleavage, which prevents the utilization in purification of disulfide bonds containing recombinant proteins. In this study, we demonstrated that the unpaired cysteine residues in Npu DnaE tend to form disulfide bonds, and contributed to the reduction of the cleavage under non-reducing conditions. This redox trap can be disrupted by site-directed mutation of these unpaired cysteines. The results further indicated that the position 28 and 59 may play certain roles in the correct folding of the active conformation. Copyright © 2018 Elsevier Inc. All rights reserved.

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