Characterization of polycationic amino acids fusion systems for ion-exchange purification of cyclodextrin glycosyltransferase from recombinant Escherichia coli.
Fusion proteins with charged polycationic amino acid tails were constructed for the purpose of simple ion-exchange purification with high purity. A number of positively charged lysine and arginine tails were fused to the C-terminus of cyclodextrin glycosyltransferase (CGTase) derived from Bacillus macerans and expressed in Escherichia coli. The ionic binding forces provided by the tails allowed the selective recovery of CGTase from recombinant E. coli cell extracts, while CGTase by itself could not bind to the cation exchanger at neutral pH. The type of amino acids used and the length of the tail directly affected the purification factors. Most intracellular proteins of E. coli adsorbed on the cation exchanger could be removed by washing with 400 mM NaCl solution at pH 7.4, suggesting that a fusion partner suitable for purification purpose should be provided with high binding strength and the maintenance of adsorption by washing with NaCl solution. Among the fusion CGTases constructed, the CGTK10ase containing 10 lysine residues provided sufficiently high binding strength to allow purification to its homogeneity through simple ion-exchange chromatography.
Kweon
Dae-Hyuk
DH
School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea.
Lee
Dae-Hee
DH
Han
Nam-Soo
NS
Rha
Chan-Su
CS
Seo
Jin-Ho
JH
D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005964:Glucosyltransferases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013045:Species Specificity
D011993:Recombinant Fusion Proteins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase