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| Enhanced Stability of Ulp1 from Saccharomyces cerevisiae S288C via SpyTag/SpyCatcher-mediated Spontaneous Cyclization |
| WANG Meng-Ke*, GUO Yu-Kun*, GUO Wan-Ying, JIA Bin, ZHANG Han, GUO Yu-Jie, WANG Jiang, YANG Guo-Yu**, HAN Ying-Qian** |
| College of Animal Science and Veterinary Medicine, Henan Agricultural University / Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture, Zhengzhou 450002, China |
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Abstract Small ubiquitin-like modifiers (SUMOs) are widely used as labels for soluble expression of recombinant protein. Ubiquitin-like-specific protease 1 (Ulp1) is a major member of SUMOs proteases; However, its poor stability hampers its applications. Moreover, studies indicated that Ulp1 fragment from residues 304 to 621 displays full proteolytic activity in cleavage reactions. Here, a cyclized recombinant truncated Ulp1 (Gly304~Lys621) was constructed with SpyTag/SpyCatcher technology, it was observed that cyclization was efficiently and spontaneously completed under a variety of environmental conditions, resulted in strong stability and maintained enzyme catalytic activity and function. To objectively verify the effect of SpyTag/SpyCatcher technology on recombinant truncated Ulp1 stability, 3 proteases were designed in this study, including recombinant truncated Ulp1-His6 (rtUlp1), SpyTag-rtUlp1-SpyCatcher-His6 (crtUlp1) and Spy-TagΔDA-rtUlp1-SpyCatcherΔEQ-His6 (lrtUlp1). The results showed that crtUlp1 exhibited higher levels of thermostability and alkali-tolerance than the other 2 variants. Additionally, the optimal temperature of the circular variant was 5 ℃ higher than that of rtUlp1 and 10 ℃ higher than that of the linear variant. These results indicated that SpyTag/SpyCatcher-mediated cyclization increased the thermodynamic and pH stability of rtUlp1 and suggested that this technology represent a promising and effective strategy for enhancing rtUlp1 stability.
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Received: 11 August 2019
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Corresponding Authors:
**twgjl@163.com; haubiochem@163.com
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