A GFP complementation system for monitoring and directing nanomaterial mediated protein delivery to human cellular organelles

Abstract

Protein-based therapeutics are gaining importance for their biocompatibility and activity toward specific targets. When these targets are intracellular, it is critical to deliver biomolecules to sites in the cell cytoplasm while retaining biomolecule activity in the complex cellular milieu. However, intracellular protein delivery is not viable unless accompanied by an active uptake mechanism or carrier mediated delivery. Moreover, once entry into the cell is achieved, detection of the biomolecule requires laborious techniques that lack real-time measurement. We have developed a fluorescence-based complementary protein delivery sensing system using split green fluorescence protein (GFP(1-10) and GFP(11)) fragments, which can be used as an indicator for protein delivery and retention of activity, and as a means to pinpoint subcellular localization. We demonstrate in vitro localized delivery by expressing the GFP(11) fragment onto the mitochondrial outer membrane of human cells, and using a model carrier (15 nm silica nanoparticles) to deliver GFP(1-10) and image trafficking and mitochondrial localization of protein delivery. Our results indicate that nanoscale materials can be used as protein carriers for targeting cell constituents including functional molecules, signaling pathways, and organelles. We envision that this GFP complementation system is ideally suited for directing nanoparticle-based delivery of drugs and other bioactive molecules into subcellular locations within cells, which can impact protein-protein interactions, signal transduction pathways, and organelle function in vitro within the context of high-throughput screening protocols.

Split Green Fluorescent Proteins: Scope, Limitations, and Outlook

“…For example, Kamiyama et al (53) used GFP11 as a multimerization scaffold to recruit the transcriptional activation domain, VP64, and enhance gene expression. With protein-based therapeutics in mind, Bale et al (6) targeted nanoparticles to specific organelles in live cells. As imaging tools, these split FP pairs help visualize endogenous localization patterns and dynamics of proteins in live cells both with standard and super-resolution microscopy (33, 49, 51, 53, 77, 105).…”

Cellular imaging by targeted assembly of hot-spot SERS and photoacoustic nanoprobes using split-fluorescent protein scaffolds

“…Both fragments, including synthetic versions of the M3 peptide, spontaneously and irreversibly self-assemble in solution to form a fully folded GFP with a mature peptide-chromophore 49 , 50 . This bimolecular complementation system has been used to target nanomaterials in cells 49 , 51 and to form protein nanostructures 52 . Here, we show that, when grafted on different metal colloids, FP fragments from GFP and its yellow (YFP) and cyan (CFP) spectral variants effectively guide the self-assembly of NPs into activatable SERS clusters and trigger the autocatalytic maturation of the FP chromophore within plasmonic hot spots that are homogenously seeded by a precise positioning of fully folded FPs at interfacial nanogaps.…”

Split‐GFP: SERS Enhancers in Plasmonic Nanocluster Probes

“…Both fragments, including synthetic versions of the M3 peptide, spontaneously and irreversibly assemble in solution to form a fully folded GFP enclosing a mature chromophore. This biomolecular complementation system has been used for cell imaging, single molecule detection in live animals, cell targeting of nanomaterials, and as a building block for supramolecular scaffolding of protein nanostructures . Here, we use these sGFP fragments to drive the formation of stable nanogaps between AuNPs and to study how nanogap seeding by the assembly of sGFP fragments influences the nanophotonic properties of a variety of AuNP clusters.…”