期刊
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
卷 209, 期 -, 页码 1001-1019出版社
ELSEVIER
DOI: 10.1016/j.ijbiomac.2022.04.078
关键词
Schizosaccharomyces pombe; Ubiquitin-like-specific protease; E. coli; Catalytic domain; SUMO; Fed-batch culture
资金
- Department of Biotechnology, Govt. of India [BT/PR30505/BBE/117/290/2018]
- CSIR
- ICMR
This study identified and characterized two novel SUMO proteases, SpUlp1 and SpUlp2, from Schizosaccharomyces pombe. The catalytic domains of these proteases were able to efficiently cleave the SUMO fusion tag from proteins, making them potential candidates for large scale protein production.
Small ubiquitin-related modifier (SUMO) proteins are efficiently used to target the soluble expression of various difficult-to-express proteins in E. coli. However, its utilization in large scale protein production is restricted by the higher cost of Ulp, which is required to cleave SUMO fusion tag from protein-of-interest to generate an authentic N-terminus. This study identified and characterized two novel SUMO proteases i.e., Ulp1 and Ulp2 from Schizosaccharomyces pombe. Codon-optimized gene sequences were cloned and expressed in E. coli. The sequence and structure of SpUlp1 and SpUlp2 catalytic domains were deduced using bioinformatics tools. Protein-protein interaction studies predicted the higher affinity of SpUlp1 towards SUMO compared to its counterpart from Saccharomyces cerevisiae (ScUlp1). The catalytic domain of SpUlp1 was purified using Ni-NTA chromatography with 83.33% recovery yield. Moreover, In vitro activity data further confirmed the fast-acting nature of SpUlp1 catalytic domain, where a 90% cleavage of fusion proteins was obtained within 1 h of incubation, indicating novelty and commercial relevance of S. pombe Ulp1. Biophysical characterization showed 8.8% alpha-helices, 36.7% beta-sheets in SpUlp1SD. From thermal CD and fluorescence data, SpUlp1SD T-m was found to be 45 ?. Further, bioprocess optimization using fed-batch cultivation resulted in 3.5 g/L of SpUlp1SD production with Y-P/X of 77.26 mg/g DCW and volumetric productivity of 205.88 mg/L/h.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据