Research Of Quantitative And High-throughput Screening System Based On Halo-Tag Technology

Halo-Tag Protein named Haloalkane dehalogenases,it is evolved dehalogenase of Rhodococcus rhodochrous.This enzyme can remove the halogen substituents from the haloalkanes,Through the replacement of dehalogenase's activity center ,to make alkyl residues can be formed covalent bond effectively with Halo-Tag protein.Halo Tag protein can very efficiently and steadily combined with ligands through covalent bond,affinity adsorb with magnetic beads or other mediums for purification soluble protein from E. coli or mammalian cells.According to Halo-Tag technology,as model based on fatty acid synthase of Mycobacterium tuberculosis,designed out serious mtFabH target small molecular compounds with Halo-Tag ligand.Used Halo-Tag and Pull-Down technology to identify and certify whether if target compounds can combin with mtFabH protein.The results showed:1,Restriction enzymes analysis of recombinant plasmid pET-32a-mtFabH and purification analysis of recombinant protein His-Trx-mtFabH results showed target gene mtFabH had inserted successfully to the prokaryotic expression vector of pET-32a (+),and obtained higher priority recombinant protein through purifying.2,Restriction enzymes analysis of recombinant plasmid pGEX-Halo-Tag and purification analysis of recombinant protein GST-Halo-Tag results showed target gene Halo-Tag had inserted successfully to the prokaryotic expression vector of pGEX-3X,and obtained higher priority recombinant protein through purifying.3,Through His-Tag Pull-Down technology to confirm whether if the interaction which target compounds of fatty acid synthase of Mycobacterium tuberculosis with Halo-Tag ligand and target protein.The results of the experiments is consistent with active of target compounds.According to the above experiments results proved Halo-Tag technology can be application in research of quantitative and high-throughput screening.