A bioengineered recombinant spider silk provides targeted G-protein-coupled receptor activation

Spider silks are renowned for exceptional mechanical properties and as promising biomaterials. Here, we introduce a recombinant chimeric protein (W 2 C ma2 ap-55) composed of two aciniform silk repetitive units (W 2 ), a major ampullate silk non-repetitive C-terminal domain (C ma2 ), and the human peptidic G-protein-coupled receptor (GPCR) ligand apelin-55 (ap-55). Ap-55 is an endogenous apelin receptor (AR) ligand, regulating various natural and pathological processes and with noted potential for therapeutic targeting. W 2 C ma2 ap-55 expressed in Escherichia coli proved amenable to both film and fiber formation, with the protein lacking ap-55 (W 2 C ma2 ) used as a control throughout. Films and fibers formed from W 2 C ma2 ap-55 were recognized by an anti-apelin antibody, in contrast to W 2 C ma2 , confirming ap-55 accessibility and intactness. Compared to W 2 C ma2 fibers, W 2 C ma2 ap-55 fibers exhibited comparable extensibility alongside enhanced strength and toughness. Films prepared from both W 2 C ma2 ap-55 and W 2 C ma2 were non-cytotoxic to HEK 293A cells stably transfected with the AR. Growth of these cells on W 2 C ma2 ap-55 films increased ERK phosphorylation relative to either W 2 C ma2 or non-silk substrate conditions, consistent with ap-55-induced AR activation. Cell attachment was also observed on the surface of W 2 C ma2 ap-55 fibers. W 2 C ma2 ap-55 is thus a promising engineered protein capable of supporting cell growth and eliciting GPCR signaling. Statement of Significance Spider silks form protein-based fibers renowned for withstanding high force and extension before breaking and for potential in biomedical application, with use of natural spider silks as sutures dating back thousands of years. Here, we introduce an engineered spider silk protein with an extension targeting a specific human cell surface receptor, the apelin receptor. This protein forms transparent films and strong and flexible fibers, supporting growth and attachment of human cells. The functionalized silk also promotes signaling in cells with the apelin receptor, while cells without it are unaffected. These new silk materials thus have enhanced functionality, allowing targeted cell signaling in the laboratory setting with potential for future application in tissue culture or biomedical device development.