Prokaryotic Expression, Purification Of Recombinant Lysostaphic Protein And Initial Study On It's Biological Activities

Staphylococcus aureus(S. aureus) is a major trouble for the most common nosocomial and community acquired infections. The emergence of antibiotic resistant organisms has been attributed to over use of broad spectrum antibiotics in humans. The antibiotic resistant staphylococcal strains are not susceptible to conventional antibiotics. At present,the vancomycin (glycopeptide) represents the last resort for the treatment of multidrug resistant staphylococci. But the strong noxious property of vancomycin limits its clinical application. Furthermore,some cases have been reported which reduced susceptibilities or resistant to vancomycin since 1996. In view of the urgent present situation, how to find a more effective medicine to control the infection, simultaneously reducing the medicine toxicity and preventing the drug resistance has become the most difficult problem in the world.Lysostaphin brings the hope to treat the multidrug resistant staphylococci. It's a zinc metalloproteinase extracted from Staphylococcus simulans that degrades the cell wall of almost all known staphylococcal species. Other Gram-positive and Gram-negative bacteria are not susceptible to this enzyme. The target of the lysostaphin activity is the pentaglycine interpeptide bridges of the unique staphylococcal peptidoglycan, where the enzyme cleaves the Gly–Gly bond. Unlike antibiotics, which interfere with the growth of the bacteria, lysostaphin is highly effective in lysing S. aureus cells in all metabolic states (growing, resting or heat killed). Moreover, the bacteria is not easy to emerge resistance to lysostaphin. The unique biological activity of lysostaphin presents numerous possibilities for applications of this enzyme to be a new antistaphylococcal agent. However, although its antimicrobial properties appeared promising, the lack of an efficient source of this protein, difficulties in its purification stopped further development of lysostaphin as a therapeutic agent. Until 1990s, along with the development of genetic engineering technology and protein purification method, some expression system were established for the recombinant lysostaphin, but still had some problems needed further to optimize:①The recombinant lysostaphin has no biologic activity or the biologic activity is very low, the enzyme activity of some recombinant protein is 10-16 times lower than natural lysostaphin;②The yield is very small;③The method of protein purification is troublesome, the time is long, the purification process causes the biological activity to lose. The expression protein is only suitable for the crystallography research in the laboratory;④The biological activity research is only limiting to the assay of the enzyme activity unit;⑤The spatial structure and the enzyme molecule properties of lysostaphin is still not clear . These problems cause many difficulties for people to study the security, the validity and the stability of lysostaphin which uses in the human body . In this thesis, the following experiments are conducted:Firstly, reconstructed the prokaryotic expression vector and optimizing expression target protein: The target gene was selective amplified by PCR using pRG recombinant plasmid as source of the lysostaphin gene, digested with Nde1and Xho1 restriction endonucleases and cloned into correspondingly digested expression vector pET-42a(+) to generate the recombinant plasmid pET42lys, The lysostaphin gene was confirmed by DNA sequencing. The pET system is the most powerful system yet developed for the cloning and expression of recombinant proteins in E. coli. Target protein expression is induced by providing a source of T7 RNA polymerase in the host cell. Another important benefit of this system is its ability to maintain target genes transcriptionally silent in the uninduced state. Target genes are initially cloned using hosts that do not contain the T7 RNA polymerase gene, thus eliminating plasmid instability due to the production of proteins potentially toxic to the host cell. Then the plasmid was transferred into an expression host(E.coli BL21(DE3)plysS) containing a chromosomal copy of the T7 RNA polymerase gene under lacUV5 control, the target protein expression is induced by the addition of IPTG to the bacterial culture. Optimization of the expression conditions revealed that 6h induction and growth at 25℃and the IPTG concentration is 1.0mmol/L . The target protein product is comprise 40% of the total cell protein and expressed mainly as inclusion bodies, only a small quantity as soluble fraction. The main reason for forming inclusion body may be overexpression .Secondly, the purification, renaturation and immunoblotting of recombinant lysostaphic protein. The protein was purified by immobilized metal ion affinity chromatography(IMAC). The advantage is that the mental ion ligand has a good stability and a big adsorption quantity, low cost and short purification time , But sometimes the nonspecific adsorption is occurred. The purification effect of soluble protein is not ideal, the purity is only 80%, Optimization purification condition for soluble protein will be studyed further. The purity of inclusion body was >95%. The concentration of soluble protein is 1151.53 ug/ml,inclusion body is 1869.54ug/ml. The yield is 15.34mg/L of shake flask induced culture . Dialysis was used for the inclusion body renaturation. The recombinant lysostaphic protein was verified by immunoblotting. Purified lysostaphic protein was responsed to anti-His.Tag monoclonal antibody, the molecular weight of recombinant lysostaphic protein was found to be approximately 27kDa as reported earlier. Thirdly, evaluated the bacteriocidal activity of recombinant lysostaphic protein in vitro. The purified protein had superactive antibacterial activities to Staphylococcus aureus (ATCC25923 and clinical isolates) and MRSA (clinical isolates) determined by plate method. The minimal inhibitory concentrations (MIC) and the minimal bactericidal concentrations (MBC) of the protein to the strains were 0.003～0.78μg/ ml and 0.39～3.125μg/ ml, whereas 3.9μg/ml of vancomycin was required to attain the same degree of inhibition. Candida albicans (clinical isolates) , pneumonococcus (clinical isolates) and aeruginosus Bacillus (clinical isolates) and other clinical isolates weren't susceptible to this enzyme. The antibacterial activities of recombinant lysostaphic protein is superior to Oxacillin, Clindamycin and Vancomycin. The specific activity( 10150U/mg) was found to be comparable to the protein reported in the literature.We found that the purified inclusion body had a good antibacterial activity and even better than soluble protein,but renatured inclusion body had no antibacterial activity. This is discrepancy with the traditional theory. The latest study reveal that some micromolecule heat shock protein may be help inclusion body keep its natural stereochemical structure and activity. The renatured inclusion body had no antibacterial activity in our research,the possibility is that we may be don't find the optimization renaturation condition and will study in the future.Finally, evaluated the factors affeced the enzyme activity of recombinant lysostaphic proteint: At present , the key point of applying the genetic engineering technology expression biological activity polypeptide is that whether the peptide still maintains the good biological activity when the acid, the alkali and temperature of environment changes . We have analyzed the effect of pH, substrate concentration, incubation temperature and time course to the enzymatic properties of recombinant lysostaphic proteint. The rate of recombinant lysostaphic protein activity was linear throughout the assay when the incubation mixture was incubated from 1 to 15min. The activity was found to be linear upto the substrate concentration of the OD of 0.9 (OD600) . The range of optimum pH for the recombinant lysostaphic protein was found to be 3.0–4.0. At pH 3.0-6.0, the recombinant protein activity was stable , this result shows the good acidproof performance of the protein but differ in reported ones. The recombinant lysostaphic protein was found to be most active at 50℃when the standard reaction mixture was incubated at different temperatures from 20℃to 60℃, where as most of the literature quotes it as 45℃- 48℃In conclusion, we have constructed the prokaryotic expression vector pET42lys successfully. The recombinant plasmid was transferred into an expression host and obtained a good yields of recombinant protein. The recombinant lysostaphic protein exhibits its superactive antibacterial activities to S. aureus cells. Our findings will bring our research work to be promising.