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Substrate specificity and biochemical characteristics of an engineered mammalian chondroitinase ABC

Research output: Contribution to journalArticlepeer-review

Philippa Warren, James Fawcett, Jessica Kwok

Original languageEnglish
JournalACS Omega
Accepted/In press30 Mar 2021

Documents

  • Warren et al 2021

    Warren_et_al_2021.pdf, 1.13 MB, application/pdf

    Uploaded date:21 Apr 2021

    Version:Final published version

    Licence:CC BY-ND

King's Authors

Abstract

Chondroitin sulphate proteoglycans inhibit regeneration, neuroprotection, and plasticity following spinal cord injury. The development of a second-generation chondroitinase ABC enzyme, capable of being secreted from mammalian cells (mChABC), has facilitated the functional recovery of animals following severe spinal trauma. The genetically modified enzyme has been shown to efficiently breakdown the inhibitory extracellular matrix surrounding cells at the site of injury while facilitate cellular integration and axonal growth. However, the activity profile of the enzyme in relation to the original bacterial chondroitinase (bChABC) has not been determined. Here, we characterise the activity profile of mChABC and compare it to bChABC, both enzymes having been maintained in physiologically relevant conditions for the duration of the experiment. We show that this genetically modified enzyme can be secreted reliably and robustly in high yield from a mammalian cell line. The modifications made to the cDNA of the enzyme have not altered the functional activity of mChABC compared to bChABC ensuring that it has optimal activity on chondroitin sulphate-A, with an optimal pH at 8.0 and temperature at 37°C. However, mChABC shows superior thermostability than bChABC ensuring the recombinant enzyme operates with enhanced activity over a variety of physiologically relevant substrates and temperatures then the widely used bacterial alternative without substantially altering its kinetic output. The determination that mChABC can function with greater robustness under physiological conditions than bChABC is an important step in the further development of this auspicious treatment strategy towards a clinical application.

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