Discovery and Identification of a Novel Tag of HlyA60 for Protein Active Aggregate Formation in .

The strategy of active aggregation tag fusion expression with target proteins can solve the problems of restricted expression, inefficient purification, and laborious immobilization faced in the production of recombinant proteins in . We localized a novel active aggregation peptide HlyA60 from the hemolysin A secretion system, which can effectively induce aggregate formation with satisfactory protein activities in after fusion expression with the protein of interest. Based on structural prediction and surface properties, the process of active aggregation of HlyA60 through electrostatic interactions and hydrophobic interactions was analyzed. To investigate the potential application of HlyA60 as an efficient aggregation tag, it was fused with acetyl xylan esterase and lipase A, separately. The resulting fusion proteins demonstrated active aggregation rates of 97.6 and 66.7%, respectively, leading to 1.9-fold and 1.7-fold increases in bacterial density at the end of fermentation. The AXE-HlyA60 fusion protein, which exhibited superior performance, was subjected to purification and immobilization. It was able to achieve column-free purification with an impressive 98.8% recovery and in situ immobilization; the immobilization enabled 30 cycles of reactions to take place with 85% residual activity maintained. Our findings provide a novel tool for efficiently producing recombinant proteins in .