Investigation of a Redox Trigger in Spider Silk Assembly
Poster Number
11A
Format
Poster Presentation
Research or Creativity Area
Natural Sciences
Abstract
Bulletproof vests and body armor represent outstanding materials made from Kevlar. Spider silk, specifically dragline silk, has been recently shown to outperform Kevlar. Dragline silk has many functional uses for spiders, ranging from locomotion to web construction. To make advances with synthetic spider silk production, a better understanding of the natural molecular process that governs spider silk assembly is needed. Some theories suggest that the transformation of spider silk proteins from a liquid-crystal phase into a solid fiber is triggered via a pH change during spider silk extrusion. Currently, our laboratory is exploring the potential for a different trigger that involves changes in the redox state (oxidation-reduction reactions). To initiate testing of this hypothesis, we created a prokaryotic expression vector carrying the cDNA encoding cysteine-rich protein 4 (CRP4), one of the main constituents of dragline silk, for production of the recombinant CRP4 in bacteria. The CRP4 cDNA was amplified by PCR, then ligated into the expression vector pCRIIBlunt. After insertion of the cDNA into the expression vector, we confirmed the presence of the CRP4 cDNA by restriction digestion and agarose gel electrophoresis, followed by DNA sequence analysis. Following confirmation of the CRP4 cDNA sequence, the pCRIIBluntII-CRP4 vector was transformed into bacteria and the recombinant protein was induced and purified using Ni-NTA affinity chromatography. Expression and purification of recombinant CRP4 was monitored by proteomics and mass spectrometry, along with SDS-PAGE analysis and silver staining. The purified recombinant CRP4 protein was shown to form large molecular weight aggregates, supporting a potential role of self-ligation that was dependent upon the oxidation state. Collectively, these results provide evidence that self-polymerization of CRP4 might be redox controlled.
Location
Don and Karen DeRosa University Center (DUC) Poster Hall
Start Date
27-4-2024 10:30 AM
End Date
27-4-2024 12:30 PM
Investigation of a Redox Trigger in Spider Silk Assembly
Don and Karen DeRosa University Center (DUC) Poster Hall
Bulletproof vests and body armor represent outstanding materials made from Kevlar. Spider silk, specifically dragline silk, has been recently shown to outperform Kevlar. Dragline silk has many functional uses for spiders, ranging from locomotion to web construction. To make advances with synthetic spider silk production, a better understanding of the natural molecular process that governs spider silk assembly is needed. Some theories suggest that the transformation of spider silk proteins from a liquid-crystal phase into a solid fiber is triggered via a pH change during spider silk extrusion. Currently, our laboratory is exploring the potential for a different trigger that involves changes in the redox state (oxidation-reduction reactions). To initiate testing of this hypothesis, we created a prokaryotic expression vector carrying the cDNA encoding cysteine-rich protein 4 (CRP4), one of the main constituents of dragline silk, for production of the recombinant CRP4 in bacteria. The CRP4 cDNA was amplified by PCR, then ligated into the expression vector pCRIIBlunt. After insertion of the cDNA into the expression vector, we confirmed the presence of the CRP4 cDNA by restriction digestion and agarose gel electrophoresis, followed by DNA sequence analysis. Following confirmation of the CRP4 cDNA sequence, the pCRIIBluntII-CRP4 vector was transformed into bacteria and the recombinant protein was induced and purified using Ni-NTA affinity chromatography. Expression and purification of recombinant CRP4 was monitored by proteomics and mass spectrometry, along with SDS-PAGE analysis and silver staining. The purified recombinant CRP4 protein was shown to form large molecular weight aggregates, supporting a potential role of self-ligation that was dependent upon the oxidation state. Collectively, these results provide evidence that self-polymerization of CRP4 might be redox controlled.
