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Bioprocess optimization for the overproduction of catalytic domain of ubiquitin-like protease 1 (Ulp1) from S. cerevisiae in E. coli fed-batch culture

Author:
Babbal,, Adivitiya,, Mohanty, Shilpa, Khasa, Yogender Pal
Source:
Enzyme and microbial technology 2019 v.120 pp. 98-109
ISSN:
0141-0229
Subject:
EDTA (chelating agent), Escherichia coli, Saccharomyces cerevisiae, active sites, additives, biopharmaceuticals, bioprocessing, bioreactors, catalytic activity, cost effectiveness, culture flasks, glycerol, histidine, octoxynol, plasmids, proteinases
Abstract:
The exploitation of SUMO (small ubiquitin-related modifier) fusion technology at a large scale for the production of therapeutic proteins with an authentic N-terminus is majorly limited due to the higher cost of ScUlp1 protease. Therefore, the cost-effective production of Saccharomyces cerevisiae Ulp1 protease catalytic domain (402–621 aa) was targeted via its cloning under strong T7 promoter with and without histidine tag. The optimization of cultivation conditions at shake flask resulted in ScUlp1 expression of 195 mg/L in TB medium with a specific product yield of 98 mg/g DCW. The leaky expression of the ScUlp1 protease was controlled using the chemically defined minimal medium. The Ni-NTA affinity purification of ScUlp1 was done near homogeneity using different additives (0.1% Triton X-100, 0.01 mM DTT, 0.02 mM EDTA and 1% glycerol) where a product purity of ∼95% with a recovery yield of 80% was obtained. The specific activity of purified ScUlp1 was found to be 3.986 × 10⁵ U/mg. The ScUlp1 protease successfully cleaved the SUMO tag even at 1:10,000 enzyme to substrate ratio with high efficacy and also showed a comparable catalytic efficiency as of commercial control. Moreover, the in vivo cleavage of SUMO tag via co-expression strategy also resulted in more than 80% cleavage of SUMO fusion protein. The optimization of high cell density cultivation strategies and maintenance of higher plasmid stability at bioreactor level resulted in the ScUlp1 production of 3.25 g/L with a specific product yield of 45.41 mg/g DCW when cells were induced at an OD600 of 132 (63.66 g/L DCW).
Agid:
6191521