Refolding and characterization of a yeast dehydrodolichyl diphosphate synthase overexpressed in Escherichia coli.
Dehydrodolichyl diphosphate synthase (DDPPs) catalyzes the sequential condensation of isopentenyl diphosphate with farnesyl diphosphate to synthesize long-chain dehydrodolichyl diphosphate, which serves as a precursor of glycosyl carrier in glycoprotein biosynthesis in eukaryotes. To perform kinetic and structural studies of DDPPs, we have expressed yeast DDPPs using Escherichia coli as the host cell. Thioredoxin and His tag were utilized to increase the solubility of the recombinant protein and facilitate its purification using Ni-nitrilotriacetic acid (NTA) column. The protein was overexpressed in E. coli but mostly existed in pellet in the absence of detergent. The low quantity of soluble DDPPs was purified using Ni-NTA, Mono Q anion-exchange, and size-column chromatographies. The protein in the pellet was solubilized with 7 M urea and purified using Ni-NTA under denaturing condition. The protein refolding was achieved via the stepwise dialysis to remove the denaturant in the presence of 6 mM beta-mercaptoethanol. Detergent n-octyl-beta-d-glucopyranoside and Triton X-100 increased the solubility of the DDPPs so that refolding can be performed at higher protein concentration. Alternatively, on-column refolding was carried out in a single step to obtain the active protein in large quantities. beta-Mercaptoethanol and Triton were both required in this quick refolding process. The kinetic studies indicated that the soluble and refolded DDPPs have comparable activities (k(cat) = 2 x 10(-4) s(-1)). Unlike its bacterial homologue, undecaprenyl diphosphate synthase, yeast DDPPs activity was not enhanced by Triton.
Chang
S Y
SY
Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei, 11529, Taiwan.
Tsai
P C
PC
Tseng
C S
CS
Liang
P H
PH
D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D004288:Dolichol Phosphates / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006639:Histidine / Q000378:metabolism; D002479:Inclusion Bodies / Q000378:metabolism; D007700:Kinetics; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013917:Thrombin / Q000378:metabolism; D014170:Transformation, Genetic; D015003:Yeasts / Q000201:enzymology*
D003902:Detergents; D004288:Dolichol Phosphates; D011993:Recombinant Fusion Proteins; D006639:Histidine; D019883:Alkyl and Aryl Transferases; C051389:dehydrodolichyl diphosphate synthetase; D013917:Thrombin