Recombinant Expression and Characterization of CRISP3 in Dragline Spider Silk

Abstract

Spider silk has been of a high interest to scientists due to its unique and diverse mechanical properties. The combination of high resilience, extensibility, and tensile strength makes spider silk an ideal material for biomedical, military, and industrial applications. Black widow spiders have seven glands that each produce a unique type of silk dedicated to different biological purposes. Of the seven silk types, our studies focused on dragline silk, a silk type produced by the major ampullate gland. Spiders use dragline silk primarily for locomotion and prey capture. Previously, only two spidroins, MaSp1 and MaSp2, have been identified as major components of dragline silk. However, recent proteomic analysis using nanoLC-MS/MS analysis with an Orbitrap Fusion™ Tribrid™ mass spectrometer has identified at least seven proteins, including members of the Cysteine-Rich Protein Family (CRP1, CRP2, and CRP4), CRISP3, fasciclin, and 2 uncharacterized proteins. In order to characterize the structure of CRISP3, we amplified the CRISP3 cDNA from a DNA library prepared from silk-producing glands using Polymerase Chain Reaction (PCR), followed by insertion of the cDNA into a prokaryotic expression vector. Following uptake of the plasmid by bacteria, cells were added in LB supplemented with ampicillin and induced to express the recombinant protein. His-tagged CRISP3 protein was purified using nickel-resin affinity chromatography and its identity confirmed by performing an in-solution tryptic digestion, followed by MS/MS analysis. In addition to performing in-solution tryptic digestions on elution fractions from the purification, we separated proteins in the elution fraction using SDS-PAGE analysis, followed by in-gel tryptic digestion and mass spectrometry. A better understanding of the molecular properties of CRISP3 will help advance the development of synthetic silk fibers that more closely resemble natural silk