A simplified method to remove fusion tags from a xylanase of Bacillus sp. HBP8 with HRV 3C protease.

Highlights • The xylanase can be detected as a soluble form when fused with MBP and Nus tags. • Small ratio of cells expressing HRV3C protease removed the tags of the fusion proteins efficiently with co-fermentation and post-fermentation methods. • The ratio of co-fermentation is smaller. • The tags were cleaved precisely and endogenous protease of E. coli had no obvious non-specific degradation to the target protein. Abstract Human rhinovirus 3C protease (HRV 3C protease) is commonly used as a tool to remove fusion tags from recombinant proteins in gene engineering due to its distinguished specificity and high activity at low temperature. This paper is aimed to simplify the strategy of removing epitope tags from target proteins with HRV 3C protease. Fusion proteins composed of a xylanase from Bacillus sp. HBP8 (xylHB) and double tags (MBP/Nus and 6×His, with an HRV 3C protease recognition site between them) were applied as substrates. To perform the cleavage and purification, strains expressing HRV 3C protease and the substrates were mixed before (co-fermentation method) or after (post-fermentation method) inducing with IPTG, followed by cell disruption and incubation at 4℃, overnight for cleavage. The soluble cytoplasmic fraction was added to Ni-NTA resin to recover the cleaved target protein. Because the process was carried out in the cell lysate, it was named as cell lysate purification system based on HRV 3C protease (CLP3C). Our data indicated small number of cells expressing HRV 3C protease was enough to remove the fusion tags efficiently with both co-fermentation and post-fermentation methods. More importantly, the tags were cleaved precisely with no obvious non-specific degradation to the target protein. Hence, active xylanase was recovered easily with this strategy. [ABSTRACT FROM AUTHOR]