Shim Kyu-Woong, Kim Dong-Hyun
Department of Life and Nanopharmaceutical Sciences and Department of Pharmaceutical Science, Kyung Hee University, 1, Hoegi, Dongdaemun-ku, Seoul 130-701, Republic of Korea.
Protein Expr Purif. 2008 Apr;58(2):222-8. doi: 10.1016/j.pep.2007.11.014. Epub 2007 Dec 8.
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.
能够降解糖胺聚糖（GAGs）的酶有助于揭示GAGs的生物学作用、结构和机制。我们从从人类肠道分离出的嗜粪拟杆菌HJ-15的基因组文库中克隆了能够降解硫酸软骨素A和C的软骨素酶AC。软骨素酶AC基因的探针（1.4 kb）是根据使用从嗜粪拟杆菌HJ-15纯化的软骨素酶AC的两个内部氨基酸序列产生的引物的PCR产物制备的。使用该探针，从包含用HindIII消化的2.5-4.5 kb DNA片段的pKF3载体基因文库中筛选出一个4 kb的软骨素酶AC阳性DNA片段。克隆的软骨素酶AC的氨基酸序列与肝素黄杆菌的氨基酸序列显示出41%的同源性。克隆的软骨素酶AC基因在表达载体pET-26b+的T7启动子下在大肠杆菌BL21(DE3)中表达，并使用His结合柱色谱法进行纯化。表达的软骨素酶AC能有效降解硫酸软骨素A和C。
