Cloning of Chondroitinase ABC from Bacteroides stercoris HJ-15, a Human Intestinal Anaerobic Bacterium

Glycosaminoglycans (GAGs), a group of linear, acidic polysaccharides composed of repeating disaccharide units of galactosamine (or glucosamine) and uronic acid (typically iduronic acid), are found in the extracellular matrix of intestinal cells [3]. GAGs have numerous biological functions, including mechanochemical switching and growth regulation [6, 7, 16]. One GAG, chondroitin sulfate, is classified into three types based on structure; chondroitin sulfate A, dermatan sulfate (chondroitin sulfate B), and chondroitin sulfate C. Chondroitinase ABC, which can degrade chondroitin sulfates A, B, and C, has been purified from the gastrointestinal bacterial species such as Proteus vulgaris [12] and Bacteroides stercoris HJ-15 [1, 5] and soil bacterial species such as Flavobacterium heparinum [16], and the genes encoding chondroitinase ABC have been cloned from P. vulgaris [12], Bacteroides thetaiotaomicron [13], Flavobacterium heparinum [16], and Yersinia pseudotuberculosis [8]. Although the native HJ-15 chondroitinase ABC has been purified, the gene has not yet been cloned. Therefore, here, we attempted to clone the chondroitinase ABC gene from HJ-15 to characterize the biochemical and genetic properties of chondroitinase ABC. The chondroitinase ABC gene of HJ-15 was cloned based on two internal amino acid sequences of the purified enzyme [5]. PCR was performed using primers csl ABC-1 and csl ABC-2 at an annealing temperature of 52C for 30 cycles. An approximately 670-bp PCR product was amplified, which was labeled with DIG (Roche, Switzerland) and used as a hybridization probe. To determine the full-length nucleotide sequence, positive clones from the genomic DNA library screened twice. In the first and second screenings, the genomic library was prepared by digestion with HindIII and KpnI (Takara, Japan), respectively. A portion of the digested chromosomal fragments (2.5−4.5 kbp) was extracted and ligated into the pKF3 vector (Takara), which was then transformed into E. coli TH2 competent cells (Takara). Positive clones were screened by Southern blotting and confirmed by DNA sequencing. To express the cloned chondroitinase ABC, the fulllength chondroitinase ABC gene from B. stercoris HJ-15 The gene encoding chondroitinase ABC from a genomic library of Bacteroides stercoris HJ-15, which was isolated from human feces, was cloned. The cloned gene consisted of 3,090 bp and was predicted to encode a 1,029−amino-acid protein. The B. stercoris chondroitinase ABC gene was not homologous to other chondroitinase ABC genes; however, its amino acid sequence showed 71% homology to that of Bacteroides thetaiotaomicron. The gene was cloned in the pET-26b+ expression vector and expressed under the T7 promoter in Escherichia coli BL21(DE3). The purified recombinant chondroitinase ABC degraded chondroitin sulfates A, B, and C.