Overexpression of arylsulfate sulfotransferase as fusion protein with glutathione S-transferase.
A procedure has been developed for the overexpression and purification of milligram quantities of the Klebsiella K-36 arylsulfate sulfotransferase (ASST). The structural gene was amplified by means of a polymerase chain reaction (PCR) technique and inserted into the plasmid vector pGEX-3X. The plasmid pGEX-100, carrying the Klebsiella K-36 astA structural gene under the control of the Escherichia coli tac promoter, was transformed into the E. coli strain BL21 (DE3). The ASST was produced in E. coli as a fusion with glutathione S-transferase. Conditions for protein production, isolation on glutathione Sepharose 4B, and Xa cleavage to generate active ASST were developed. The purification yielded approximately 0.7 mg of pure enzyme per liter of bacterial culture. Kinetic analysis of the overexpressed enzyme indicated that it had kinetic properties almost the same as those of the enzyme purified from Klebsiella K-36 cells. The purification procedure was very rapid and is suitable for obtaining considerable amounts of enzyme at a relatively high yield compared with its purifying method from the culture of the Klebsiella K-36 strain.
Baek
M C
MC
College of Pharmacy, Seoul National University, Korea.
Choi
K H
KH
Oh
T G
TG
Kim
D H
DH
Choi
E C
EC
D000595:Amino Acid Sequence; D015239:Arylsulfotransferase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D005798:Genes, Bacterial; D005982:Glutathione Transferase / Q000096:biosynthesis; D007700:Kinetics; D007709:Klebsiella / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D015239:Arylsulfotransferase; C090707:arylsulfate sulfotransferase