The Expression,Purification And Activity Study Of The Cytoplasmic Domain In Arl S From Staphylococcus Aureus

Staphylococcus aureus is a typical gram-positive bacteria, and is one of the main pathogenic bacteria of mankind. It can cause the wound of purulent infections and the infection of internal organs. S. aureus may cause pneumonia, endocarditis and sepsis, and other diseases. As a matter of fact,it may bring a reduction of the immune ability, even to death.The pathogenic mechanism of S. aureus is bound up with the secretion of various extracellular toxins and enzymes. When S. aureus cause infection to host cells, it may express a series of virulence factors, which regulated by the complex network composed of multiple genes. The arl two-component signal transduction system regulates the expression of a variety of virulence factors directly or indirectly. The arl system consists of the histidine kinase protein ArlS and the response regulator protein ArlR. ArlS can catch the extracellular signals and transfer signals to intracellular, and give rise to the phosphorylation of ArlR, then open the arl two-component signal transduction system. Therefore, the arl two-component signal transduction system and the histidine kinase protein ArlS are regard as drug targets which can provide treatments for S. aureus infections. This will be of great importance in both reducing or inhibiting the virulence factors and controlling fections caused by S. aureus.In this study, the unrestricted cloning method was used to amplify target gene. Then the recombinant plasmids pProEX-HTa-arls and pProEX-HTa-arlr, were transfered into DH5αcompetent cell, and induced the expression of target proteins. We used different kinds of isolation technologies such as the metal ion affinity chromatography, the ion exchange chromatography and the gel filtration chromatography, to express and purify proteins. The production of ArlR can reach 25 mg with about 98% purity from one liter culture medium,and the production of ArlS can reach 15 mg with about 90% purity from one liter culture medium. The circular dichroism detection results showed that the purified ArlR completed with natural secondary structure. In order to ensure the purifed ArlS has kinase activity, we used Kinase-Glo Luminescent Kinase Assay to measure the kinase activity of ArlS. In vitro phosphorylation results showed that ArlS had kinase activity and had the ability ofautophosphorylation. Then the phosphorylated ArlS transfers the phosphate groups to the response regulator protein ArlR. The recombinant plasmids pProEX-HTa-ArlS_CAG418A and pProEX-HTa-ArlS_CAG420A were constructed by the method of site-direted mutation, then used the metal ion affinity chromatography technologies to express and purify proteins, and detected the kinase activity of ArlS_CAG418A and ArlS_CAG420A. The results showed that ArlS_CAG418A and ArlS_CAG420A did not have the kinase activity. This illustrated that the 418 and420 amino acid residues play an important role in the autophosphorylation process of ArlS.