title: Affinity-based release of chondroitinase ABC from a modified methylcellulose hydrogel.
abstract: Chondroitinase ABC (ChABC) is a promising therapeutic for spinal cord injury as it can degrade the glial scar that is detrimental to regrowth and repair. However, the sustained delivery of bioactive ChABC is a challenge requiring highly invasive methods such as intra-spinal injections, insertion of intrathecal catheters, or implantation of delivery vehicles directly into the tissue. ChABC is thermally unstable, further complicating its delivery. Moreover, there are no commercial antibodies available for its detection. To achieve controlled release, we designed an affinity-based system that sustained the release of bioactive ChABC for at least 7days. ChABC was recombinantly expressed as a fusion protein with Src homology domain 3 (SH3) with an N-terminal histidine (HIS) tag and a C-terminal FLAG tag (ChABC-SH3). Protein purification was achieved using a nickel affinity column and, for the first time, direct quantification of ChABC down to 0.1nM was attained using an in-house HIS/FLAG double tag ELISA. The release of active ChABC-SH3 was sustained from a methylcellulose hydrogel covalently modified with an SH3 binding peptide. The rate of release was tunable by varying either the binding strength of the SH3-protein/SH3-peptide pair or the SH3-peptide to SH3-protein ratio. This innovative system has the potential to be used as a platform technology for the release and detection of other proteins that can be expressed using a similar construct.
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 1A1, Canada.
authors.forename: Malgosia M
authors.initials: MM
authors.lastname: Pakulska
authors.forename: Katarina
authors.initials: K
authors.lastname: Vulic
authors.forename: Molly S
authors.initials: MS
authors.lastname: Shoichet
mesh_terms: D019765:Chondroitin ABC Lyase / Q000737:chemistry* / Q000235:genetics; D002846:Chromatography, Affinity; D003692:Delayed-Action Preparations / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D020100:Hydrogels; D008747:Methylcellulose / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D018909:src Homology Domains
chemical_list: D003692:Delayed-Action Preparations; D020100:Hydrogels; D010455:Peptides; D011993:Recombinant Fusion Proteins; D008747:Methylcellulose; D019765:Chondroitin ABC Lyase
keywords: Affinity release; Chondroitinase ABC; Controlled release; ELISA; SH3 protein–peptide binding partners