Preliminary Studies On Expression,Purification,and Structure Of The Plasmid Encoded Outer Membrane Protein T Of Avian Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli(APEC)is the primary pathogen causing avian colibacillosis.The main clinical manifestations of this disease include Escherichia coli sepsis,pericarditis,perihepatitis,and air sacculitis.It has caused huge economic losses to the poultry industry.APEC has many virulence factors,among which,outer membrane protease is a key virulence factor that plays a central role on the host-pathogen interface.Outer membrane protein T(OmpT)is a proteolytic enzyme existing in the outer membrane of E.coli and belongs to the Omptin outer membrane protease family,which may play a role in the adhesion process during bacterial infection.The OmpT encoded by the chromosome of Escherichia coli K12 is the first protein to be resolved in the Omptin family.The OmpT consists of 10 antiparallel ?-sheets that form a hollow barrel-like structure,extending from the lipid bilayer of the outer membrane to the outside of the cell.Our laboratory has found that APEC E058 strain(02 serotype)possesses both OmpT(cOmpT)encoded by the chromosomal gene(compT)and OmpT(pOmpT)encoded by the ColV plasmid gene(pompT).The amino acid homology of cOmpT and pOmpT was as high as 73.6%.At present,the study on cOmpT is relatively sufficient,but that on pOmpT is rare.The preliminary research data of our laboratory has showed that cOmpT can hydrolyze protamine.but pOmpT can't hydrolyze protamine although it plays an important role in the pathogenesis of APEC.Thus,it is necessary to conduct crystallographic studies on pOmpT in order to find the potential structural difference between pOmpT and cOmpT.In this study,we constructed a pOmpT(without signal peptide)recombinant protein expression strain E.coli BL21(DE3)pET30a-pompT,then obtained pOmpT protein in the form of inclusion body after induction and expression.The pOmpT protein had been denatured and renatured in vitro,unfortunately,self-hydrolysis occurred after gel filtration chromatography,which is unfavourable to subsequent crystallization experiments.According to relevant reports,cOmpT can avoid self-hydrolysis after K217G site-mutation.Therefore,we constructed BL21(DE3)pET30a-pompT(K217G)and BL21(DE3)pET30a-pompT(K217G)-His expression strains,then expressed pOmpT(K217G)and pOmpT(K217G)-His respectively.The pOmpT(K217G)-His could be subjected to the new detergent which was more suitable for crystallization when purified through the nickel column.Finally,we purified the target proteins with gel filtration chromatography to prepare for the crystallization experiment.After X-ray diffraction,we found that the crystals of pOmpT(K217G)were all salt crystals,while most of the crystals of pOmpT(K217G)-His were protein crystals.Therefore,we used pOmpT(K217G)-His for further crystallization optimization screening.After optimization,the stable crystals of 7 A at MemMeso-17 were observed Our extensive exploration of the optimal crystallization conditions for pOmpT laid a fundamental basis for resolving the detailed crystal structure of pOmpT,which is necessary to clarify the biological function of pOmpT.Additionally,we constructed pET30a-compT-His and pET30a-compT(K237G)-His recombinant plasmids containing cOmpT signal peptides,we also constructed pET30a-pompT-His and pET30a-pompT(K237G)-His recombinant plasmids containing pOmpT signal peptides,and then transformed these four plasmids into BL21(DE3)respectively.Using E058 and BL21(DE3)pET30a as controls,we tested the ability of these six strains to hydrolyze protamine.The results showed that cOmpT without site mutation can hydrolyze protamine,while cOmpT with K237G site mutation(K217G site mutation of the fragment of cOmpT without signal peptide)can not.Whether with site mutation or not,pOmpT does not have this ability.The results indicated that the K237 site not only plays a key role in the stabilization of cOmpT and pOmpT,but also in the enzymatic activity of cOmpT to hydrolyze protamine.