Cloning and expression of chondroitinase AC from Bacteroides stercoris HJ-15.
Enzymes that degrade glycosaminoglycans (GAGs) can help reveal the biological roles, structure, and mechanisms of GAGs. We cloned chondroitinase AC, which can degrade chondroitin sulfates A and C, from the genomic library of Bacteroides stercoris HJ-15 isolated from human intestine. The probe (1.4 kb) for the chondroitinase AC gene was prepared from the PCR product of the primers produced using two internal amino acid sequences of chondroitinase AC purified from B. stercoris HJ-15. Using this probe, a chondroitinase AC-positive, 4 kb DNA fragment was selected from pKF3 vector gene libraries containing 2.5-4.5 kb DNA fragments digested with HindIII. The amino acid sequence of the cloned chondroitinase AC showed 41% homology to that of Flavobacterium heparinum. The cloned chondroitinase AC gene was expressed under the T7 promoter of the expression vector, pET-26b+, in Escherichia coli BL21(DE3) and purified using His bind column chromatography. The expressed chondroitinase AC potently degraded chondroitin sulfates A and C.
Shim
Kyu-Woong
KW
Department of Life and Nanopharmaceutical Sciences and Department of Pharmaceutical Science, Kyung Hee University, 1, Hoegi, Dongdaemun-ku, Seoul 130-701, Republic of Korea.
Kim
Dong-Hyun
DH
D000595:Amino Acid Sequence; D001439:Bacteroides / Q000201:enzymology*; D001483:Base Sequence; D002808:Chondroitin Lyases / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D015698:Genomic Library; D006801:Humans; D007422:Intestines / Q000382:microbiology; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013379:Substrate Specificity
D002808:Chondroitin Lyases