[No authors listed]
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 (402-621âaa) 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 195âmg/L in TB medium with a specific product yield of 98âmg/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, 0.01âmM DTT, 0.02âmM EDTA and 1% glycerol) where a product purity of â¼95% with a recovery yield of 80% was obtained. The specific activity of purified ScUlp1 was found to be 3.986âÃâ105âU/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 3.25âg/L with a specific product yield of 45.41âmg/g DCW when cells were induced at an OD600 of 132 (63.66âg/L DCW).
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