Production and purification of recombinant human laminin-111 for pre-clinical testing in a murine model of laminin-α2 deficient congenital muscular dystrophy
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Authors
Smith, Deryan
Issue Date
2017
Type
Thesis
Language
en_US
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Abstract
Laminin α2-deficient congenital muscular dystrophy (LAMA2-CMD) is a fatal genetic neuromuscular disease that currently has no cure. Patients with LAMA2-CMD suffer from muscle wasting, respiratory issues, and neurological effects until eventual death at a premature age due to loss of the adult isoforms of laminin, laminin 211/221, from the basal lamina. Previous research in the Burkin lab has shown effective treatment of the dyW mouse model of LAMA2-CMD through successful substitution of these adult laminin isoforms using injections of the mouse embryonic laminin isoform, laminin-111. What has not been explored in these studies is whether the dyW mouse model could also be successfully treated with a recombinant human form of laminin-111, as human patients would require treatment with the human isoform. To determine whether human laminin-111 could be used to treat the LAMA2-CMD mouse model, recombinant human laminin-111 (rhLAM-111) was produced in Chinese hamster ovary (CHO) cells for extraction and purification to be later used for pre-clinical testing in the dyW mouse model. Our results show rhLAM-111 purification was unsuccessful, indicating heparin affinity chromatography, gel filtration, immobilized metal affinity chromatography (IMAC), and phosphocellulose columns were not efficient or optimized for successful purification and concentration of rhLAM-111. These results indicate the need for a different purification strategy in order to produce enough pure laminin for further research examining the effectiveness of rhLAM-111 in treating the dyW mouse model of LAMA2-CMD
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Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 United States