| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | E. coli strain BL21 (DE3) (Vazyme, Nanjing, China) preserved in this laboratory was used for recombinant plasmid amplification and protein expression. The plasmid pET21b (Novagen, Madison, WI, USA) harboring a C-terminal 6×His tag was used for protein purification and was preserved in this laboratory. The pET21b-SUMO-rtUlp1-His plasmid was synthesized by GenScript (Nanjing, China). SpyTag and SpyCatcher sequences were synthesized by Sangon Biotech (Shanghai, China). The plasmid (pET21b-His6-Cherry-TEV-SUMO-sfGFP) used for expression (substrate protein) was constructed in this laboratory. Both restriction endonucleases and Page Ruler unstained or prestained protein ladders were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Primer STARTM DNA polymerase and T4 DNA ligase were purchased from TaKaRa Bio (Dalian, China). The SanPrep column plasmid miniprep kit, DNA gel extraction kit, and PCR product purification kit were purchased from Sangon Biotech (Shanghai, China). High-affinity Ni-NTA resin was obtained from GenScript. All other chemicals were from Sigma-Aldrich (Shanghai, China). | Get A Quote |
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 e... More
