Rapid purification of HRV3C and TEV proteases using polyhistidine and polylysine tags

In vivo enzymatic digestion of HRV 3C protease cleavage sites-containing proteins produced in a silkworm-baculovirus expression system

“…In this regard, the quality and quantity of VP15 products are both required for fundamental and applied studies. Upon mixing and incubating the cell lysates of E. coli expressing GST-HRV3C or GST-3Csite-VP15 on ice for 4 h, GST-3Csite-VP15 was digested efficiently, and the resulting products as predicted can be clearly seen and verified from SDS-PAGE analysis ( Figure 2 B), suggesting the GST-3C was active in E. coli lysate as compared to previous studies from active MBP-fused 3C [ 17 , 18 ].…”

“…Previously, we designed an in vivo covalent binding platform between SpyTag/SpyCatcher (ST/SC)-tagged protein partners in silkworm-BEVS, termed SpyBEVS [ 29 ]. The current result in successful recognition and digestion between 3C proteinase and its recognition residuals is another practical example of protein expression and engineering using the BEVS platform, which can be further applied to other proteinases, such as TEV proteinase [ 17 ]. The current platform is also helpful to produce and purify fusion tag-free proteins or protein complexes of interest as needed in antigen and vaccine development.…”

“…However, the efficiency of in vitro protease-based reactions is condition-dependent (buffer, temperature, etc.) [ 17–19 ]. Moreover, the second purification to remove free tags and proteases with further buffer exchange procedures is very time-consuming and possibly results in a significant loss of POIs, either in the amount or overall activity.…”