Influencing the degradation rate of recombinant spider silk in the presence of matrix metalloproteinases

Coekin, Thomas (2021) Influencing the degradation rate of recombinant spider silk in the presence of matrix metalloproteinases. PhD thesis, University of Nottingham.

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Understanding the degradation behaviour of extracellular matrix (ECM) scaffolds is essential for predicting and advancing wound healing. Spider-silk based proteins are one type of biomaterial with the potential to be used as a matrix to improve would healing. In addition to good biocompatibility and low-pyrogenicity, silk-based biomaterials have displayed the capacity for controlled degradation, a characteristic that is investigated in this study.

In silico studies took target sequences of human matrix metalloproteinase 2 and 9 (MMP2 and MMP9) and compared them to sequences of silk major ampullate spidroin 1 (MaSp1) termini of spider genera: Araneus, Argiope, Cyrtophora, Dolomedes, Euprosthenops and Nephila and the recombinant synthetic mini-spidroin 4RepCT to identify locations for potential mutations to influence the protein’s degradation. Proteolytic degradation has been carried out in vitro with dragline silk fibres of a range of species from distantly related spider families namely Cyrtophora citricola, Dolomedes fimbriatus, Pisaura mirabilis, Pholcus phalangioides and Nephila madagascarensis to confirm the predicted degradation seen from in silico studies.

Based on the MaSp1 of Euprosthenops australis, 4RepCT was recombinantly expressed and degraded by human neutrophil elastase (ELNE), MMP2 and MMP9. From the MMP profiles of 4RepCT, 14 mutation sites were identified, with a final seven being carried forward to experimentation due to location within the structured spidroin. Of these seven, two mutations located near the thrombin cleavage site and within the structured C-terminus were successfully expressed in DL41 and BL21 E. coli, respectively.

Successfully expressed mutant spidroins were subjected to MMP2 (>1000 pmol/min/μg protein) and MMP9 (>1300 pmol/min/μg protein) concentrations that were approximately 10% of that typically seen in chronic wounds. Spidroins with a mutation in the amorphous region of the spidroin gene increased the degradation rate, degrading 1 mg/mL protein in 30 minutes with both proteases, whereas mutations within the structured C-terminus did not degrade in the same way. This suggests that while introducing target sites can influence the rate of degradation, the sites must be accessible to the protease in question.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Thomas, Neil
Goodacre, Sara
Keywords: Biomaterial, Matrix metalloproteinase, Degradation, 4RepCT, Scaffold, Biodegradation, Target sequences, Recombinant, Spidroin, Synthetic
Subjects: Q Science > QD Chemistry
Faculties/Schools: UK Campuses > Faculty of Science > School of Chemistry
Item ID: 64470
Depositing User: COEKIN, Thomas
Date Deposited: 31 Jul 2021 04:40
Last Modified: 31 Jul 2021 04:40

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