Immobilization of Ulp1 protease on NHS-activated Sepharose: a useful tool for cleavage of the SUMO tag of recombinant proteins
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To fabricate an active and stable enzyme through covalent immobilization, a Ubl-specific protease (Ulp1) was used to cleave small ubiquitin-like modifier (SUMO) fusion proteins.
We immobilized Ulp1 on N-hydroxysuccinimide (NHS)-activated Sepharose with a coupling efficiency of 1.7 mg/ml. The immobilized Ulp1 maintains 95% substrate-cleavage ability and significantly enhances pH and thermal stability, especially can withstand pH of 10.5. Besides resistance against some small molecules, the immobilized Ulp1 can tolerate 15% (v/v) DMSO and 20% (v/v) ethanol. It can be reused for more than 15 batch reactions with 90% activity retention. This provides a fast purification system to quickly obtain cleaved recombinant proteins with 95% purity from cell lysates with the application of immobilized Ulp1.
Ulp1 used in immobilization form is a potentially useful tool for cleavage of SUMO-tagged proteins and may reduce time and cost of protein purification.
KeywordsEnzymatic cleavage Extreme pHs tolerance Immobilization Thermal stability Ubl-specific protease (Ulp1)
This work is supported by the Natural Science Foundation of China (31270831 and 30970630), the Outstanding Youth Science Foundation of Chongqing (cstc2011jjjq10003), the Program for New Century Excellent Talents in University (NCET-08-0912), the Fundamental Research Funds for the Central Universities (XDJK2016C158) and the Doctoral Fund of Southwest University (XJKJXM003338).
Supplementary Fig. 1—Different immobilization strategies for Ulp1.
Supplementary Fig. 2—DMSO inhibits the degradation of SUMO.
Supplementary Fig. 3—The influence of small molecules on the activity of Ulp1.
Supplementary Fig. 4—Storage stability of free and immobilized Ulp1 at 30 °C.
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