Diversity Of Glycosyl Transferase Structural Biology Research

Objectives Will contain His-N-end tag label of the nature of the substrate miscellaneous generic mango C-glycosyl transferase Mi CGTb preliminarily by Ni-column purification,and then through multistage HPLC separation protein with high purity.By screening of different length protein crystallization conditions(including temperature,buffer,p H,protein concentration,the precipitation agent,etc.),determine suitable for the study of the structure of protein sequences,get no substrate(donor and receptor)combined with glycosyl transferase crystal structure,and determine the active center.Methods 1 Will source for mango known sequence of C-glycosyl transferase Mi CGTb genes by designing primers amplification connected to p ET28 carrier,its C-glycosyl transferase Mi CGTb containing N-end His-tag label and TEV enzyme loci,build p ET28-Mi CGTb carrier expressed in RIPL host,end of IPTG concentration is 0.2 m M,30? induced expression,the Ni-column in preliminary purification,again by anion exchange column purification,protein separation of high purity in the end.2 Will source for mango known sequence of C-glycosyl transferase Mi CGTb gene by protein disorder degree of system analysis,respectively,the N-and C-the amputation of seven and five amino acids,after by designing primers amplification connected to p ET28 carrier,cloning truncated Cafter the glycosyl transferase Mi CGTb(JMi CGTb)containing His-N-end tag label and TEV enzyme loci,build p ET28-JMi CGTb carrier expressed in RIPL host,end of IPTG concentration is 0.2 m M,30 ? induced expression,the Ni-column in preliminary purification,again by anion exchange column purification,protein separation of high purity in the end.3 Build p ET28-JMi CGTb carrier expressed in B834 express the host,and obtain protein containing rare element selenium,the experimental data obtained for anomalous scattering experiment.4 Use the screen kit screening protein crystallization conditions,at the beginning of the early screening kits for Hampton?and?,CRYO1 and 2 two product kits,make up for each other between the crystallization of the lack of conditions.Screen and screen are used at the beginning of the 16 th hole hanging drop method,after a lot of early screening,after screening and optimum conditions finally got relatively good JMi CGTb crystalsResults 1 Optimization of C-glycosyl transferase Mi CGTb expression strains,try different expression of the host,obtain high expression of C-glycosyl transferase Mi CGTb strains 2 through different purification methods to obtain high purity of C-glycosyl transferase Mi CGTb protein 3 Get C-glycosyl transferase Mi CGTb single crystal and XRay diffraction dataConclusions 1 Obtain high expression of truncated C-glycosyl transferase MiCGTb expression strains 2 To obtain high purity of truncated C-glycosyl transferase Mi CGTb protein 3 Get a truncated C-glycosyl transferase Mi CGTb protein crystal 4 Get a set of C-6 Emmanuel glycosyl transferase Mi CGTb protein single crystal X-Ray diffraction scan data...