Site-Specific Labeling of Surface Proteins on Living Cells Using Genetically Encoded Peptides that Bind Fluorescent Nanoparticle Probes

Rocco, Mark A.; Burns, Andrew; Kostecki, Jan; Doody, Anne M.; Wiesner, Ulrich; DeLisa, Matthew P.

doi:10.1021/bc9000118

Cited by 11 publications

(10 citation statements)

References 46 publications

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“…Therefore, we next investigated whether outer membrane proteins could be N glycosylated. In a manner similar to the strategy described above for MBP, we engineered a GT acceptor sequence into the second extracellular loop of the ␤-barrel outer membrane protein OmpX from E. coli (44). Cells expressing OmpX-GT in the presence of the pgl locus produced glycosylated OmpX, whereas OmpX-GT expressed in cells carrying the pgl mut locus showed no detectable glycosylation (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The plasmid pBAD24-OmpX-GT was created by inserting the GT sequence between the KpnI and SpeI sites of plasmid pBAD24-OmpX*-His (where * indicates a mutated form of OmpX containing a cloning site for peptide insertion into loop 2) (44) such that the GT peptide tag was positioned between the serine residues at positions 53 and 54 of extracellular loop 2 of OmpX. This transmembrane loop is able to tolerate short peptide insertions without affecting the surface expression of OmpX (44). A GQSGQ linker that flanked the KpnI and SpeI sites was also introduced.…”

Section: Methodsmentioning

confidence: 99%

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Production of Secretory and Extracellular N-Linked Glycoproteins inEscherichia coli

Fisher

Haitjema

Guarino

et al. 2011

Appl Environ Microbiol

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118

108

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The Campylobacter jejuni pgl gene cluster encodes a complete N-linked protein glycosylation pathway that can be functionally transferred into Escherichia coli. In this system, we analyzed the interplay between N-linked glycosylation, membrane translocation and folding of acceptor proteins in bacteria. We developed a recombinant N-glycan acceptor peptide tag that permits N-linked glycosylation of diverse recombinant proteins expressed in the periplasm of glycosylation-competent E. coli cells. With this "glycosylation tag," a clear difference was observed in the glycosylation patterns found on periplasmic proteins depending on their mode of inner membrane translocation (i.e., Sec, signal recognition particle [SRP], or twin-arginine translocation [Tat] export), indicating that the mode of protein export can influence N-glycosylation efficiency. We also established that engineered substrate proteins targeted to environments beyond the periplasm, such as the outer membrane, the membrane vesicles, and the extracellular medium, could serve as substrates for N-linked glycosylation. Taken together, our results demonstrate that the C. jejuni N-glycosylation machinery is compatible with distinct secretory mechanisms in E. coli, effectively expanding the N-linked glycome of recombinant E. coli. Moreover, this simple glycosylation tag strategy expands the glycoengineering toolbox and opens the door to bacterial synthesis of a wide array of recombinant glycoprotein conjugates.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Production of Secretory and Extracellular N-Linked Glycoproteins inEscherichia coli

Fisher

Haitjema

Guarino

et al. 2011

Appl Environ Microbiol

Self Cite

118

108

View full text Add to dashboard Cite

show abstract

“…Through the use of combinatorial phage display techniques, researchers have been able to design short peptides, which bind specifically to particular inorganic surfaces [39]–[42]. These peptides can be included in a given protein to give it a moiety that will bind specifically to inorganic surfaces such as silica [43]. In contrast to such specialized proteins, we find that the binding of our model protein to silica is nonspecific, but is by no means trivial.…”

Section: Discussionmentioning

confidence: 99%

Modeling Pressure-Driven Transport of Proteins Through a Nanochannel

Carr

Comer

Ginsberg

et al. 2011

IEEE Trans. Nanotechnology

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Reducing the size of a nanofluidic channel not only creates new opportunities for high-precision manipulation of biological macromolecules, but also makes the performance of the entire nanofluidic system more susceptible to undesirable interactions between the transported biomolecules and the walls of the channel. In this manuscript, we report molecular dynamics simulations of a pressure-driven flow through a silica nanochannel that characterized, with atomic resolution, adsorption of a model protein to its surface. Although the simulated adsorption of the proteins was found to be nonspecific, it had a dramatic effect on the rate of the protein transport. To determine the relative strength of the protein–silica interactions in different adsorbed states, we simulated flow-induced desorption of the proteins from the silica surface. Our analysis of the protein conformations in the adsorbed states did not reveal any simple dependence of the adsorption strength on the size and composition of the protein–silica contact, suggesting that the heterogeneity of the silica surface may be a important factor.

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“…This is very important to limit the degree of nonspecific interactions and to enhance specific binding by the target sites or internalization by cells that overexpress the target receptors. 2,[45][46][47][48] A proper and fine control of functionalization can also yield long-life systems in which the nanoparticle head-off by the RES (Reticulo-Endothelial System) is delayed.…”

Section: Prodimentioning

confidence: 99%

Dye-doped silica nanoparticles as luminescent organized systems for nanomedicine

et al. 2014

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The ability to find synergic solutions is the core of scientific research and scientific advancement. This is particularly true for medicine, where multimodal imaging and theranostic tools represent the frontier research. Nanotechnology, which by its very nature is multidisciplinary, has opened up the way to the engineering of new organized materials endowed with improved performances. In particular, merging nanoparticles and luminescent signalling can lead to the creation of unique tools for the design of inexpensive, hand-held diagnostic and theranostic kits. In this wide scenario, dye-doped silica nanoparticles constitute very effective nanoplatforms to obtain efficient luminescent, stable, biocompatible and targeted agents for biomedical applications. In this review we discuss the state of the art in the field of luminescent silica-based nanoparticles for medical imaging, starting with an overview of the most common synthetic approaches to these materials. Trying to rationalize the presentation of this extremely multifaceted and complex subject, we have gathered significant examples of systems applied in cancer research, also discussing those that take a multifunctional approach, including theranostic structures. Nanoprobes designed for applications that do not include cancer are a minor part, but interesting achievements have been published and we present a selection of these in the subsequent section. To conclude, we propose a debate on the advantages of creating chemosensors based on luminescent silica nanoparticles. This is far from easy but is a particularly valuable goal in the medical field and therefore subject to extensive research worldwide.

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Site-Specific Labeling of Surface Proteins on Living Cells Using Genetically Encoded Peptides that Bind Fluorescent Nanoparticle Probes

Cited by 11 publications

References 46 publications

Production of Secretory and Extracellular N-Linked Glycoproteins inEscherichia coli

Production of Secretory and Extracellular N-Linked Glycoproteins inEscherichia coli

Modeling Pressure-Driven Transport of Proteins Through a Nanochannel

Dye-doped silica nanoparticles as luminescent organized systems for nanomedicine

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