Effects Of The N-Terminal Of Protease HtrAw On The Growth Of Escherichia Coli

Thermophilic protease HtrAw is a serine protease derived from thermophile Brevibacillus sp.WF146 and belongs to the subtilisin family.In the previous experiments,Dr.Zhu Fengtao found that when protease HtrAw expressed in E.coli BL21(DE3),it will not only increase the host bacteria viable cells significantly,but also influence the cell morphology,ultimately determined that the N-terminal peptide N74 of protease HtrAw played an important role.Based on this study,we investigated the effect of N74 on the growth of Escherichia coli.Thermophilic protease WF146 is an extracellular serine protease secreted by Brevibacillus sp.WF146.For its thermal stability and activity,it can not only be used for theoretical research of enzyme structure and functional,but also has potential application value in the industry.In order to make full use of the recombinant protein N74 to improve the biomass of E.coli,we co-expressed N74 and protease WF146△S which was lack of the signal peptide.We found that there was no significant difference in the expression of protease WF146 between co-expression of N74+WF146△S and individual expression of WF146△S,and the growth status of co-expressed N74+WF146△S recombinant bacteria was significantly weaker than that of individually expressed WF146△S.We speculated that in the co-expression system,the simultaneous expression of the two proteins increased the metabolic burden of the host to a great extent,so that the host bacteria could not take into account both growth and reproduction of foreign proteins.In this study,we analyzed the distribution of WF146△S in the co-expression system and the individual expression system which showed WF146△S of the extracellular was much more than the latter,indicating that the expression of recombinant protein N74 could damage host cell to a certain extent.In the industrial production,most of the protein expressed mainly in the cells,so we can take use of this feature of N74 to help some of the protein expressed in soluble form into the extracellular,thus simplifying the broken cell separation of the target protein process.Additionally,we used the green fluorescent protein gene as the reporter gene to construct the recombinant plasmid pN74-GFP.Then,we expressed the recombinant protein in E.coli BL21(DE3),and by observing the distribution of the fluorescent protein we could know the distribution of the recombinant protein N74.We found that the distribution of the recombinant protein N74 in the host bacteria was in a clear spiral,and this distribution was similar with some functional proteins which involved in the division of the host cells and have been reported earlier,we guess the expression of the recombinant protein through a certain model of action,interacting with the functional proteins,resulting in the filamentous.But the specific mechanism need to further exploration and confirmation.In this study,we explored the mechanism of recombinant protein N74 influenced the growth of Escherichia coli to help us understand the multiple factors that affect the growth of Escherichia coli and have some reference value for industrial production.