Study On Expression, Purification And Function Of Cas Protein In M. Tuberculosis

Recently, in the genomes of the most bacteria (40%) and almost all of the archaea (90%), it was discovered that a group of clustered regularly interspaced short palindromic repeats (CRISPR). For bacteria, CRISPR is an adaptive immune mechanism, which is used to defense against bacteriophages and viruses so that bacteria could survive in the harsh natural environmental. It is because the CRISPR system provides the bacteria with phage-specific immunity, so it will have a broad prospect in both biomedicine and bioindustry.Currently, tuberculosis is one of the highest date rate of single pathogenic bacterium infectious diseases which induced by the single pathogenic bacterium. China is one of the high tuberculosis burden countries and the population of tuberculosis patients ranks second in the world, second only to India. In China, the population of dying from tuberculosis is about250,000every year, which is more than the twice of the sum of all kinds of infectious diseases. However, in recent years, the high drug-resistance strains of Mycobacterium tuberculosis increased gradually in all over the world, which is because Mycobacterium tuberculosis prone to generate drug resistance, and even caused the tuberculosis outbreak. At the same time, it made that the current drug has been insufficient to restrain Mycobacterium tuberculosis growth. So, it is greatly important to investigate deeply to the pathogenic mechanism and the immune mechanism of Mycobacterium tuberculosis, the pathogenic bacterium of tuberculosis. The CRISPR system is one of the important immune mechanisms for Mycobacterium tuberculosis. Therefore, this study does a preliminary exploration on the CRISPR system of Mycobacterium tuberculosis.From some previous studies on certain bacteria, it was reasonable to deduce the presence of CRISPR system in Mycobacterium tuberculosis.The process of CRISPR system contains two steps. First, the phage invaded into the bacteria. The Cas protein recognized it, and integrate its DNA into the host genome. Then, in the second phage invasion, CRISPR system could identify the exogenous phage, and end the immune process by using the specific ribose endonuclease to cut the invaded phage. In this process, Casl and Cas2involved in obtaining the phage information, and Cas6protein, a ribose endonuclease which could cut CRISPR repeat RNA, plays an important role in the defense process.In this study, three recombinant expression plasmids (MBP-Rv2816, MBP-Rv2817and MBP-Rv2824) were successfully constructed, and highly expressed in the pronucleus. The target genes Rv2816, Rv2817, Rv2824corresponded to three target proteins Casl-mbp, Cas2-mbp and Cas6-mbp, respectively. Vector pPDB-His-MBP was selected as the expression plasmid, and the size of protein marker MBP was45KDa. The MBP marker in the vector could enhance the solubility of the fusion proteins, particularly the eukaryotic protein, which were over-expressed in the bacteria. The purification of protein was carried out by employing Ni2+-affinity chromatography (IMAC). The His-carrying fusion protein specifically binded to the gel matrix (i.e. the chromatography column) of IMAC. The column-binding fusion protein could be separated by the elution using a highly concentrated (400mM) imidazole buffer solution.With reference to the expression of these three target proteins, the fusion proteins Casl-mbp and Cas6-mbp depicted excellent solubility after16℃induced expression; while Cas2-mbp was severely degraded after purification. The fusion protein Cas6-mbp was used for the follow-up study, i.e. used to establish in vitro systems of Cas6-mbp severing CRISPR-repeat RNA and severing CRISPR-spacer RNA. The experimental samples were divided into two groups, which used CRISPR-repeat RNA and CRISPR-spacer RNA as the reaction substrate, respectively. While the amount of RNA maintained the same, the amounts of Cas6-mbp were designed to increase according to a concentration gradient for both groups. In this experiment, another RNA-negative group was set up as the control group. From the results, Protein Cas6appeared to be able to sever CRISPR-repeat RNA, but not able to sever CRISPR-spacer RNA.In this paper, the presence of CRISPR system in Mycobacterium tuberculosis was preliminarily interpreted. In addition, the existence of Cas1, Cas2and Cas6genes in the genome of mycobacterium tuberculosis, as well as the validity of expression purification on these genes, were examined. Among them, protein Cas6could serve as an endoribonuclease. The findings of this study may probably facilitate a further understanding on the immunological mechanism of Mycobacterium tuberculosis, so as to constitute an important reference for the biomedicine studies in the future.