Effect Of Extremely Halophilic Archaea Protein NJ7G＿0991 On The Integrity Of The Outer Membrane In Escherichia Coli BL21

NJ7G＿0991 is a protein secreted from extremely halophilic archaea Natrinema gari J7-2 through Sec-dependent pathway,which has a higher abundance in the extracellular components.The previous bioinformatic analysis found that NJ7G＿0991may have a Lol A-like structure domain.Lol A plays an important role in the lipoprotein transport localization system in Escherichia coli.Previous studies found that the cell morphology of the NJ7G＿0991 Lol A domain deletion mutant of strain J7-2,changed from rod to round.When NJ7G＿0991 was recombinant and expressed in Escherichia coli,the protein was mostly distributed in the extracellular components of the culture medium.In order to study the function of the Lol A domain,we constructed two mutants of NJ7G＿0991 lacking the signal peptide(NJ7G＿0991 Δ S)and the C-terminal domain(NJ7G＿0991ΔC),respectively,and expressed them in Escherichia coli.We found that NJ7G＿0991ΔC,like NJ7G＿0991,was expressed in large amounts in the extracellular components,while NJ7G＿0991 Δ S accumulated in the whole cell components;the strain expressed the mutant NJ7G＿0991ΔC with Lol A domain and signal peptide had a significant lower tolerance to lysozyme compared to the strain transferred into the empty plasmid p ET26 b.Most cells of BL21/NJ7G＿0991 and BL21/NJ7G＿0991ΔC were observed to be rounded after lysozyme treatment under the microscope.While the cell morphology of the strain expressed the mutant NJ7G＿0991ΔS without signal peptide did not change significantly.Indicating that the expression of NJ7G＿0991 or NJ7G＿0991 Δ C will cause damage to the outer membrane of the cell.We speculate that this two proteins may interfere with the outer membrane lipoprotein transport system in Escherichia coli,resulting in the damage to the integrity of the outer membrane,making NJ7G＿0991 and some proteins in periplasmic space be distributed outside the cell.Which means that the Lol A domain of NJ7G＿0991 may also play an important role in maintaining the integrity of the envelope structure of halophilic archaea.We constructed the co-expression plasmids(p D16 and p D61)of NJ7G＿0991 and protease WF146 using the plasmid p ETDuet-1 with dual promoters and multiple cloning sites according to the characteristics of NJ7G＿0991 causing limited damage to the outer membrane of Escherichia coli.The leaky expression system may improve the extracellular expression of the protease,which is more conducive to the collection and utilization of the target protein.The results of transparent zones showed that the transformants transformed into the co-expression plasmid of NJ7G＿0991 and WF146 may produce more extracellular WF146 than the transformants expressing WF146 alone.The leaky expression system may improve protein production efficiency in industrial production and other fields,and obtain the target protein more conveniently.It also provides new information for the maintenance mechanism of cell membrane integrity in halophilic archaea.