Expression and purification of dalcochinase, a beta-glucosidase from Dalbergia cochinchinensis Pierre, in yeast and bacterial hosts.
The coding sequence of the mature dalcochinase, a beta-glucosidase from Dalbergia cochinchinensis Pierre, was cloned and expressed in various systems. Expression in Escherichia coli resulted in an insoluble protein, which could be made soluble by co-expression with bacterial chaperonin GroESL. However, the enzyme had no activity. Recombinant expression in Pichia pastoris and Saccharomyces cerevisiae yielded an active enzyme. Dalcochinase was expressed under methanol induction in P. pastoris, since this was much more efficient than constitutive expression in P. pastoris or in S. cerevisiae. Addition of 0.5% casamino acids to the culture medium stabilized the pH of the culture and increased the protein yield by 3- to 5-folds. Insertion of a polyhistidine-tag either after the N-terminal alpha factor signal sequence or at the C-terminus failed to assist in purification by immobilized metal-ion affinity chromatography (IMAC) due to post-translational processing at both termini. A new construct of dalcochinase with an N-terminal truncation following the propeptide and eight histidine residues enabled its purification by IMAC, following hydrophobic interaction chromatography. The purified recombinant dalcochinase was apparently composed of differently post-translationally modified forms, but had kinetic properties and pH and temperature optima comparable to natural dalcochinase. The procedures reported here overcome the limitation in enzyme supply from natural sources, and allow further studies on structure-function relationships in this enzyme.
Toonkool
Prachumporn
P
Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok, Thailand.
Metheenukul
Pornphimon
P
Sujiwattanarat
Penporn
P
Paiboon
Patcharee
P
Tongtubtim
Nusra
N
Ketudat-Cairns
Mariena
M
Ketudat-Cairns
James
J
Svasti
Jisnuson
J
D000818:Animals; D004926:Escherichia coli / Q000235:genetics*; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D015003:Yeasts / Q000235:genetics*; D001617:beta-Glucosidase / Q000235:genetics* / Q000302:isolation & purification*
D011994:Recombinant Proteins; D001617:beta-Glucosidase