The Expression And Purification Of Sulfolobus Islandicus REY15A Cmr Complex

Clusters of regularly interspaced short palindromic repeat(CRISPR)is found in most archaeal(?90%)and bacterial(-40%)genomes.CRISPR sequence and CRISPR associated protein together constitutes the CRISPR-Cas system,which is called the adapted immune system of prokaryote.CRISPR-Cas system is utilized to eliminate the threat of non-self gene element like virus and plasmid.And as the fact that CRISPR-Cas9 has an outstanding performance in gene editing,research on CRISPR-Cas system gains more attention.In Sulfolobus islandicus REY15A,there are three types of CRISPR-Cas system,one kind of CRISPR type ? A system and two kinds of CRISPR III B system,of which the Cas protein complex is not well studied.To attain one of the Cas protein complex named Cmr complex,pSeSD,a shuttle plasmid of E.coli and Sulfolobus islandicus,is used to construct a vector,which is used in the expression of Cmr3 subunit of Cmr complex.When transformed into Sulfolobus islandicus REY15A mutant E233S(a uracil defective strain)and induced by arabinose,the plasmid can be used to produce His-tagged Cmr3 protein subunit.Given the fact that Cmr subunits interact with one another,Cmr complex can be purified with His tag.