Lysyl oxidase (LOX) and the four lysyl oxidase like proteins, LOXL, LOXL2, LOXL3 and LOXL4, are copper-containing amine oxidases constitute a heterogeneous family of enzymes that oxidize primary amine substrates to reactive aldehydes, catalyzing the cross-linking of extracellular matrix (ECM) proteins. LOXL2 induces epithelial-to-mesenchymal transition (EMT), which is associated with hypoxia, enhanced invasion, cancer metastasis and poorer cancer prognosis. Furthermore, upregulation of LOXL2 mRNA and/ or protein levels has been detected in undifferentiated breast, colon, esophagus and larynx carcinomas. The aim here is to create and optimize a method to produce large yields of enzymatically active recombinant LOXL2 protein from mammalian cells. Two viral transductions systems were used to transfect CHO-K1 cells to overexpress LOXL2 protein. Comparing lentivirus transduction with adenovirus transduction, it was found that adenovirus transduction expressed 2.18 fold the amount of enzymatically active LOXL2 compared to lentivirus transduction (P<0.05). The average LOXL2 yield of lentivirus and adenovirus transduction systems as calculated by BCA assay was 184.5 µg and 403 µg, respectively. The average specific LOXL2 enzymatic activity were calculated using an Amplex red assay and found to be 0.443 and 0.444 nmol/μg of LOXL2 in 30 minutes in lentivirus and adenovirus methods, respectively, with no statistically significant difference (P>0.05). Expression and purification of LOXL2 were confirmed by SDS-PAGE and Western blot. Optimizing this method to purify large yields of LOXL2 is a practical aid in revealing the exact structure and function of the LOX family of proteins.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/18311 |
| Date | 28 September 2016 |
| Creators | Mously, Eihab Abdullah |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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