| E.coli is used as a common host for heterologous expression of recombinant proteins owing to its clear genetic background,multiple tools for genetic manipulation,high compatibility,fast growth rate and easy scale-up culture.However,the production of recombinant proteins in E.coli often suffers from restricted expression,susceptibility to protease degradation and inefficient purification,and time-consuming and laborious optimization of immobilization for enzymes used in biocatalysis.The strategy of forming active inclusion bodies by fusion expression of active aggregation tags with target proteins,which has arisen in recent researches,provides a new way of solving the above problems.Active inclusion bodies display excellent performance in the production of recombinant proteins,mainly attributed to their aqueous insolubility.The two-step protein washing method mediated by this strategy,as a rapid,economical and column-free purification method,can simplify the recombinant protein purification process and substantially reduce the purification cost.At the same time,the active aggregation tag-mediated in situ immobilization can achieve comparable performance to the widely used carrier-binding immobilization.In this study,based on the ineffectiveness of the E.coli type I secretion system with mCherry as the target of secretion,the target protein was found to exist intracellularly in the form of active inclusion bodies.By comparing the combination of proteins required for different type I secretion systems,the gene hlyA218 with active aggregation function was identified,further gene truncation identified the active aggregation short peptide HlyA60.Combined with the structural prediction and surface property analysis,the active aggregation principle of HlyA60 through electrostatic interaction two-by-two attraction and hydrophobic interaction aggregation to form tubular or spherical structures was preliminarily hypothesized.After the fusion expression with enzymes,HlyA60 was capable of forming enzyme-active aggregates effectively in E.coli.In particular,the acetylxylan esterases（AXE）fusion protein AXE-HlyA60 had 97.6%active inclusion formation and 1.91 times higher bacterial density(OD600)than the control strain at the end of fermentation.Compared with other active aggregation tags reported previously,HlyA60 had better"pull-down"ability while retaining AXE enzyme activity,and substantially increased the growth rate and fermentation density of the strain.Given the insolubility of AXE-HlyA60,the column-free purification was achieved by a two-step washing method with 98.8%recovery of enzyme activity.The in situ immobilized AXE-HlyA60 achieved 30 batches of cyclic catalysis and still retained 85.8%of enzyme activity.This study identified a novel short peptide,HlyA60,which induces intracellular protein aggregation in E.coli,showing a high rate of protein aggregation and the potential to stimulate rapid growth of recombinant bacteria.The discovery of this short peptide provides a new possible option for the expansion of active aggregation protein applications. |
