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Show COLLEGE OF PHARMACY UNDERGRADUATE RESEARCH ABSTRACTS CLONING AND EXPRESSION OF CHONDROITINASE ABC FROM PROTEUS VULGARIS Orlando Antelope (April Joice) Department of Medicinal Chemistry University of Utah Glycosamioglycans (GAGs) are linear sulfated polysaccharides comprised of repeating disaccharide units of alternating a hexosamine linked to an uronic acid, which are involved in many biological processes. Galactosaminoglycans (GalAGs) are a subgroup of GAGs, which are known to inhibit the ability for a nerve to regenerate following a central nervous system injury. Chondroitinases are enzymes that depolymerize the GalAGs. The removal of GalAG chains in damaged nerve tissue by chondroitinase ABC (ChABC) promotes the recovery of neurons. This project focuses on the cloning of ChABC, which is used in the lab for further research into nerve regeneration. Using genomic DNA, a forward primer was designed to include an Xhol restriction site, and the reverse primer was designed to include a BamHI restriction site.The ChABC encoding D N A sequence is amplified from Proteus vulgaris genomic DNA by PCR and sub-cloned into the pGEM-T vector. Once confirmed by sequencing, the ChABC is removed from the pGEM-T vector by restriction digest and cloned into a pET-19b vector for expression of the recombinant enzyme. Variations of this technique will then be used for other G A G enzymes used to efficiently produce pure enzymes for research. ChABC ChABC Orlando Antelope OH ' Chondroitin Sulfate (CS) . Representation of chondroitinase A B C cleavage In the gh/cosaminogrycan chain (where R Is S03 or H). Polvmer sequences are cleaved at the pi ,4-bonds by chondroitinase ABC, generating disaccharides from the chain interior, and mono-or disaccharides from the chain terminal. fTSTTTSfeftf^wm Figure 2. Primers used tor cloning ChABC. In order to expedite cloning Into a pGEM-T vector, the forward primers were designed to incorporate an Xhol restriction site, and the reverse primers were designed with a BamHI restriction site. A spacer was also added into each primer to ensure proper cutting of the gene. XAmotficallon.OloMion ^Dkjestlon ChABC 2995 bp Laddfli | ^ ^ ^ 8 3. Genomic UNA of P. vulgaris was obtained from stock sources. PCR was facilitated using P. vulgaris genomic DNA as the template. The PCR product was then llgated Into the pGEM-T vector. Figured. PCR b used to amplify the ChABC in the pGEM-T vector. Confirmation of presence of tanjet enzyme (ChABC) II performed uibij eel electrophoresis. ChABC Is known to be 2595 bp. the gal Image Indicates the presence of D N A of "3000 bp. The eel also Indicates the presence of smaller unwanted derivative. April Joice 55 |