Part:BBa_K5185032
rtUlp1（Recombinant truncated Ubiquitin-like protease 1）
Small Ubiquitin-like Modifier (SUMO) is a ubiquitin-related protein that regulates the function of target proteins by binding to lysine residues. It can be recognized and cleaved by SUMO proteases, leaving no amino acid residues upon cleavage by SUMO tag proteases. This can increase the solubility of expressed proteins, affect the folding of C-terminal fusion proteins, and protect target peptides from degradation by proteases, making it commonly used for expressing small antimicrobial peptides. Although Ulp1 is an attractive tool for obtaining target proteins with natural N-terminal sequences, its application in cutting SUMO fusion proteins is greatly restricted due to its expensive price and complex procedures. rtUlp retains the Leu1403-Lys631 of yeast Ulp, making it smaller in molecular weight and easier to recombinantly express.
Results
A recombinant Ulp1 from Escherichia coli is produced by constructing a truncated Ulp1 strain (rtUlp1) that retains the enzyme's active center (Wang et al. 2016) and achieving soluble expression in a 400 mL flask (Fig.A). Enzyme digestion reaction under the same conditions as the purchased Ulp1 enzyme was performed. CBM3-SUMO-HNP1AWK (BBa_K5185031) was chosen as the substrate to conduct antimicrobial assay on E. coli.
Figure 1: Expression and enzyme digestion verification of rtUlp1
(a)SDS-PAGE identification of soluble expression of rtUlp1，where S represents the supernatant of cell lysate and IB represents inclusion bodies.
(b) Identification of enzymatic activity of rtUlp1.
After our expressed rtUlp1 processed CBM3-SUMO-HNP1AWK, it displayed the same inhibition zone as the CBM3-SUMO↓HNP1AWK processed by Ulp1, indicating that our expressed rtUlp1 has SUMO enzyme cutting activity. In this experiment, we took 200 mL of the fermentation broth from the 400 mL shaker flask for purification, and obtained 3 mL of rtUlp1 (concentration not tested). We then added 1 μL to successfully achieve the enzyme-mediated release of the defensin. Therefore, we believe that the cost of producing our own Ulp1 is extremely low, and it can be mass-produced for wound dressings.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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