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1.
Adv Mater ; 31(12): e1806727, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30687975

ABSTRACT

Interrogation and control of cellular fate and function using optogenetics is providing revolutionary insights into biology. Optogenetic control of cells is achieved by coupling genetically encoded photoreceptors to cellular effectors and enables unprecedented spatiotemporal control of signaling processes. Here, a fast and reversibly switchable photoreceptor is used to tune the mechanical properties of polymer materials in a fully reversible, wavelength-specific, and dose- and space-controlled manner. By integrating engineered cyanobacterial phytochrome 1 into a poly(ethylene glycol) matrix, hydrogel materials responsive to light in the cell-compatible red/far-red spectrum are synthesized. These materials are applied to study in human mesenchymal stem cells how different mechanosignaling pathways respond to changing mechanical environments and to control the migration of primary immune cells in 3D. This optogenetics-inspired matrix allows fundamental questions of how cells react to dynamic mechanical environments to be addressed. Further, remote control of such matrices can create new opportunities for tissue engineering or provide a basis for optically stimulated drug depots.

2.
J Control Release ; 165(1): 38-43, 2013 Jan 10.
Article in English | MEDLINE | ID: mdl-23142579

ABSTRACT

Hydrogels provide a highly favorable matrix for immobilizing growth factors, enzymes or cells for biomedical applications like tissue engineering, drug delivery or the treatment of metabolic diseases. In this study we describe the synthesis and characterization of a hydrogel able to degrade L-ornithine, a metabolite that is highly elevated in congenital hyperornithinemia. The hydrogel was synthesized by embedding the L-ornithine-degrading enzymes L-ornithine aminotransferase (OAT) and L-ornithine decarboxylase (ODC) into a polymer network. The network was formed from linear polyacrylamide crosslinked by heterodimers of ODC and ornithine decarboxylase antizyme (OAz). The resulting hydrogel was shown to be stable under physiological conditions and to efficiently degrade L-ornithine. The hydrogel-stabilizing ODC-OAz interactions could subsequently be dissociated by the addition of antizyme inhibitor (AzI) which resulted in the inducible dissolution of the hydrogel. This L-ornithine-degrading hydrogel that can efficiently be eliminated when its functionality is no longer required might represent a first step towards an enzyme substitution approach against hyperornithinemia.


Subject(s)
Hydrogel, Polyethylene Glycol Dimethacrylate/chemistry , Ornithine Decarboxylase/chemistry , Ornithine-Oxo-Acid Transaminase/chemistry , Ornithine/chemistry , Proteins/chemistry , Acrylic Resins/chemistry , Carrier Proteins/chemistry , Carrier Proteins/metabolism , HEK293 Cells , Humans , Hydrogel, Polyethylene Glycol Dimethacrylate/metabolism , Ornithine/metabolism , Ornithine Decarboxylase/metabolism , Ornithine-Oxo-Acid Transaminase/metabolism , Proteins/metabolism
3.
Macromol Rapid Commun ; 33(24): 2103-8, 2012 Dec 21.
Article in English | MEDLINE | ID: mdl-23079933

ABSTRACT

Metabolite-responsive hydrogels that detect pathological metabolite concentrations and react by releasing a therapeutic stimulus hold high promises in treating metabolic diseases. In this study, a hydrogel is described that discriminates between physiological and pathological concentrations of urate, the causative agent of gouty arthritis. The hydrogel is synthesized by coupling a dimeric variant of the Deinococcus radiodurans-derived urate repressor HucR to linear polyacrylamide. The protein-grafted polymer is crosslinked to form a hydrogel by a multimeric hucO DNA sequence [hucO]n specifically binding HucR. At elevated urate concentrations, HucR dissociates from [hucO]n thereby weakening the hydrogel structure and resulting in its dissolution.


Subject(s)
Acrylic Resins/chemistry , Bacterial Proteins/chemistry , Biosensing Techniques , DNA/chemistry , Hydrogels/chemistry , Uric Acid/analysis , Bacterial Proteins/biosynthesis , Bacterial Proteins/isolation & purification , Cross-Linking Reagents/chemistry , DNA/chemical synthesis , Deinococcus/chemistry , Electrophoretic Mobility Shift Assay , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Kinetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Repressor Proteins/biosynthesis , Repressor Proteins/chemistry , Repressor Proteins/isolation & purification
4.
Org Biomol Chem ; 10(33): 6629-32, 2012 Sep 07.
Article in English | MEDLINE | ID: mdl-22821135

ABSTRACT

The Cu(I)-catalyzed cycloaddition of terminal azides and alkynes (click chemistry) represents a highly specific reaction for the functionalization of biomolecules with chemical moieties such as dyes or polymer matrices. In this study we evaluate the use of bicinchoninic acid (BCA) as a ligand for Cu(I) under physiological reaction conditions. We demonstrate that the BCA-Cu(I)-complex represents an efficient catalyst for the conjugation of fluorophores or biotin to alkyne- or azide-functionalized proteins resulting in increased or at least equal reaction yields compared to commonly used catalysts like Cu(I) in complex with TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine) or BPAA (bathophenanthroline disulfonic acid). The stabilization of Cu(I) with BCA represents a new strategy for achieving highly efficient bioconjugation reactions under physiological conditions in many application fields.


Subject(s)
Alkynes/chemistry , Azides/chemistry , Copper/chemistry , Quinolines/chemistry , Catalysis , Click Chemistry/methods , Coloring Agents/chemistry , Coordination Complexes/chemistry , Cycloaddition Reaction/methods , Ligands , Lipase/chemistry , Thermoanaerobacter/enzymology
5.
Protein Expr Purif ; 66(2): 158-64, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19324091

ABSTRACT

Inducer-dependent prokaryotic transcriptional repressor proteins that originally evolved to orchestrate the transcriptome with intracellular and extracellular metabolite pools, have become universal tools in synthetic biology, drug discovery, diagnostics and functional genomics. Production of the repressor proteins is often limited due to inhibiting effects on the production host and requires iterative process optimization for each individual repressor. At the example of the Streptomyces pristinaespiralis-derived streptogramin-dependent repressor PIP, the expression of which was shown to inhibit growth of Escherichia coli BL21*, we demonstrate that the addition of the PIP-specific streptogramin antibiotic pristinamycin I neutralizes the growth-inhibiting effect and results in >100-fold increased PIP titers. The yield of PIP was further increased 2.5-fold by the engineering of a new E. coli host suitable for the production of growth-inhibiting proteins encoded by an unfavorable codon usage. PIP produced in the presence of pristinamycin I was purified and was shown to retain the antibiotic-dependent binding to its operator pir as demonstrated by a fluorescence resonance energy transfer (FRET)-based approach. At the example of the macrolide-, tetracycline- and arsenic-dependent repressors MphR(A), TetR and ArsR, we further demonstrate that the production yields can be increased 2- to 3-fold by the addition of the cognate inducer molecules erythromycin, tetracycline and As(3+), respectively. Therefore, the addition of inducer molecules specific to the target repressor protein seems to be a general strategy to increase the yield of this interesting protein class.


Subject(s)
Anti-Bacterial Agents/metabolism , Bacterial Proteins/metabolism , Escherichia coli/metabolism , Repressor Proteins/metabolism , Allosteric Regulation , Bacterial Proteins/genetics , Cell Proliferation/drug effects , Codon/genetics , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Peptide Synthases/genetics , Peptide Synthases/metabolism , Pristinamycin/pharmacology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Repressor Proteins/genetics , Streptomyces/genetics , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
6.
Nat Mater ; 7(10): 800-4, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18690239

ABSTRACT

Drug-dependent dissociation or association of cellular receptors represents a potent pharmacologic mode of action for regulating cell fate and function. Transferring the knowledge of pharmacologically triggered protein-protein interactions to materials science will enable novel design concepts for stimuli-sensing smart hydrogels. Here, we show the design and validation of an antibiotic-sensing hydrogel for the trigger-inducible release of human vascular endothelial growth factor. Genetically engineered bacterial gyrase subunit B (GyrB) (ref. 4) coupled to polyacrylamide was dimerized by the addition of the aminocoumarin antibiotic coumermycin, resulting in hydrogel formation. Addition of increasing concentrations of clinically validated novobiocin (Albamycin) dissociated the GyrB subunits, thereby resulting in dissociation of the hydrogel and dose- and time-dependent liberation of the entrapped protein pharmaceutical VEGF(121) for triggering proliferation of human umbilical vein endothelial cells. Pharmacologically controlled hydrogels have the potential to fulfil the promises of stimuli-sensing materials as smart devices for spatiotemporally controlled delivery of drugs within the patient.


Subject(s)
Delayed-Action Preparations/administration & dosage , Hydrogels/administration & dosage , Acrylic Resins/administration & dosage , Acrylic Resins/chemistry , Aminocoumarins/administration & dosage , Biocompatible Materials/administration & dosage , Biocompatible Materials/chemistry , Cell Proliferation/drug effects , DNA Gyrase/administration & dosage , DNA Gyrase/chemistry , Endothelial Cells/cytology , Endothelial Cells/drug effects , Humans , Hydrogels/chemistry , Materials Testing , Molecular Structure , Novobiocin/administration & dosage , Vascular Endothelial Growth Factor A/administration & dosage
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