Bio-orthogonal Immobilization of Fibroblast Growth Factor 2 for Spatial Controlled Cell Proliferation

Lühmann, Tessa; Jones, Gabriel Davis; Gutmann, Marcus; Rybak, Jens-Christoph; Nickel, J. Curtis; Rubini, Marina; Meinel, Lorenz

doi:10.1021/acsbiomaterials.5b00236

Cited by 35 publications

(49 citation statements)

References 40 publications

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“…The product (referred to as denatonium carboxylate) was directly coupled to the N-terminus of the PCL. The PCLs had an unnatural azido alanine at their C-terminus through which the PCL was coupled to ethinyl-functionalized polymethylmethacrylate (PMMA) microparticles through the copper(I) catalyzed variant of the azide-alkyne Huisgen cycloaddition 18 – 20 . These peptide sensors were referred to as the first generation (Fig.…”

Section: Resultsmentioning

confidence: 99%

Diagnosing peri-implant disease using the tongue as a 24/7 detector

et al. 2017

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Our ability of screening broad communities for clinically asymptomatic diseases critically drives population health. Sensory chewing gums are presented targeting the tongue as 24/7 detector allowing diagnosis by “anyone, anywhere, anytime”. The chewing gum contains peptide sensors consisting of a protease cleavable linker in between a bitter substance and a microparticle. Matrix metalloproteinases in the oral cavity, as upregulated in peri-implant disease, specifically target the protease cleavable linker while chewing the gum, thereby generating bitterness for detection by the tongue. The peptide sensors prove significant success in discriminating saliva collected from patients with peri-implant disease versus clinically asymptomatic volunteers. Superior outcome is demonstrated over commercially available protease-based tests in saliva. “Anyone, anywhere, anytime” diagnostics are within reach for oral inflammation. Expanding this platform technology to other diseases in the future features this diagnostic as a massive screening tool potentially maximizing impact on population health.

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Section: Resultsmentioning

confidence: 99%

Diagnosing peri-implant disease using the tongue as a 24/7 detector

et al. 2017

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“…[30][31] Briefly, protein expression of the fusionprotein pHisTrx-FGF-2 was induced with 0.2 mM IPTG at OD600 = 0.6 and expression was performed at 30° C at 200 rpm. After 5h cells were harvested by centrifugation and were resuspended in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM PMSF, pH 7.5) and solubilized by sonification at 4° C. After centrifugation at 100,000 g for 1h at 4° C (L8-60M Ultracentrifuge, Beckman-Coulter, Brea, CA), the supernatant containing pHisTrx tagged FGF-2 was purified by heparin-sepharose affinity chromatography using an FPLC system (Aekta Purifier, fusionprotein pHisTrx-FGF-2 was cleaved by thrombin (GE, Freiburg, Germany) with a final concentration of 1U thrombin/mg fusionprotein at 4°C overnight.…”

Section: Expression and Purification Of Murine Fgf-2mentioning

confidence: 99%

Nanomechanics on FGF-2 and Heparin Reveal Slip Bond Characteristics with pH Dependency

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Ozer

Jones

et al. 2017

ACS Biomater. Sci. Eng.

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Fibroblast growth factor 2 (FGF-2) -an important paracrine growth factor -binds electrostatically with low micro-molar affinity to heparan sulphates present on extracellular matrix proteins. A single molecular analysis served as a basis to decipher the nanomechanical mechanism of the interaction between FGF-2 and the heparan sulphate surrogate -heparin -with a modular atomic force microscope (AFM) design combining magnetic actuators with force measurements at the low force regime (10 1 -10 4 pN/s). Unbinding events between FGF-2-heparin complexes were specific and short-lived. Binding between FGF-2 and heparin had strong slip bond characteristics as demonstrated by a decrease of life-time with tensile force on the complex. Unbinding forces between FGF-2 and heparin were further detailed at different pH as relevant for (patho-) physiological conditions. An acidic pH environment (5.5) modulated FGF-2 -heparin binding as demonstrated by enhanced rupture forces needed to release FGF-2 from heparin-FGF-2 complex as compared to physiological conditions. This study provides a mechanistic and hypothesis driven model on how molecular forces may impact FGF-2 release and storage during tissue remodeling and repair.3

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“…1b ). The azide and alkyne functionalities of the selected uAA enable biorthogonal click chemistry as demonstrated by myoglobin [ 13 ], ubiquitin [ 14 ] or basic fibroblast growth factor [ 16 ] and for site-specific protein modification of the glycocalyx on living cells [ 17 ]. The formation of uAA-eGFP and biomass is monitored through the transparent bottom of microtiter plates with a screening platform constructed in-house in a modified BioLector setup [ 18 , 19 ].…”

Section: Resultsmentioning

confidence: 99%

Probing unnatural amino acid integration into enhanced green fluorescent protein by genetic code expansion with a high-throughput screening platform

et al. 2016

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BackgroundGenetic code expansion has developed into an elegant tool to incorporate unnatural amino acids (uAA) at predefined sites in the protein backbone in response to an amber codon. However, recombinant production and yield of uAA comprising proteins are challenged due to the additional translation machinery required for uAA incorporation.ResultsWe developed a microtiter plate-based high-throughput monitoring system (HTMS) to study and optimize uAA integration in the model protein enhanced green fluorescence protein (eGFP). Two uAA, propargyl-L-lysine (Plk) and (S)-2-amino-6-((2-azidoethoxy) carbonylamino) hexanoic acid (Alk), were incorporated at the same site into eGFP co-expressing the native PylRS/tRNAPylCUA pair originating from Methanosarcina barkeri in E. coli. The site-specific uAA functionalization was confirmed by LC-MS/MS analysis. uAA-eGFP production and biomass growth in parallelized E. coli cultivations was correlated to (i) uAA concentration and the (ii) time of uAA addition to the expression medium as well as to induction parameters including the (iii) time and (iv) amount of IPTG supplementation. The online measurements of the HTMS were consolidated by end point-detection using standard enzyme-linked immunosorbent procedures.ConclusionThe developed HTMS is powerful tool for parallelized and rapid screening. In light of uAA integration, future applications may include parallelized screening of different PylRS/tRNAPylCUA pairs as well as further optimization of culture conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13036-016-0031-6) contains supplementary material, which is available to authorized users.

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Bio-orthogonal Immobilization of Fibroblast Growth Factor 2 for Spatial Controlled Cell Proliferation

Cited by 35 publications

References 40 publications

Diagnosing peri-implant disease using the tongue as a 24/7 detector

Diagnosing peri-implant disease using the tongue as a 24/7 detector

Nanomechanics on FGF-2 and Heparin Reveal Slip Bond Characteristics with pH Dependency

Probing unnatural amino acid integration into enhanced green fluorescent protein by genetic code expansion with a high-throughput screening platform

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