Fol. Biol. 2021, 67, 82-89

https://doi.org/10.14712/fb2021067020082

A Preliminary Characterization of a Novel Recombinant Clostridial Collagenase Blend

Ivan Leontovyč1, T. Koblas1, Z. Berková1, K. Bittenglova2,3, A. Leontovyc4, M. Benesik5, F. Saudek3

1Laboratory for the Islets of Langerhans, Department of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
2Department of Biomedicine – Cell Biology and Pathology, First Faculty of Medicine, Charles University, Prague, Czech Republic
3Department of Diabetes, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
4Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic
5Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic

Received December 2020
Accepted April 2021

Clostridial collagenases are essential biotechnological tissue dissociation agents owing to their ability to cleave different types of collagen. Standardization of collagenase-based protocols has been hampered by impurities in products manufactured from Clostridium histolyticum. To enhance the purification process, we produced recombinant collagenase classes G and H, taking advantage of the Escherichia coli expression system. The respective gene sequences were derived from C. histolyticum and modified by addition of a C-terminal polyhistidine tag. Harvested bacteria were lysed and the collagenase protein was affinity purified using a His-tag column. The purity, identity, integrity of the eluted collagenases G and H were determined by SDS electrophoresis and Western blot. The proteolytic activity of the collagenase G and H blend (rColGH) was determined by the standard FALGPA assay. The tissue dissociation activity was verified using a standardized method for isolation of rat pancreatic islets. Biocompatibility of the blend was validated by a standardized viability assay on the isolated islets. Two batches of rColGH were produced and compared to a commercially available collagenase. Based on our results, we conclude that rColGH is a functional and non-toxic novel recombinant collagenase worth further characterization and blend optimization in order to make it a competitive commercial product.

Funding

The research was supported by a grant from the Ministry of Industry and Trade, Czech Republic, Project FV20139.

References

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