Bioinspired Pseudozwitterionic Hydrogels with Bioactive Enzyme Immobilization via pH-Responsive Regulation

Hydrogels are hydrated networks of flexible polymers with versatile biomedical applications, and their resistance to nonspecific protein adsorption is critical. On the other hand, functionalization with other biomacromolecules would greatly enhance their biotechnological potential. The aim of this research is to prepare low fouling hydrogel polymers for selective protein immobilization. Initially, hydrogels were prepared by controlling the composition ratios of 2-carboxyethyl acrylate (CA) and 2-dimethylaminoethyl methacrylate (DMAEMA) monomers in an N,N-methylene-bis-acrylamide (NMBA) cross-linked free radical polymerization reaction. This series of hydrogels (C1D9 to C9D1) were then analyzed by X-ray photoelectron spectroscopy (XPS) and dynamic laser scattering to confirm the actual polymer ratios and surface charge. When the composition ratio was set at CA:6 vs DMEAMA:4 (C6D4), the hydrogel showed nearly neutral surface charge and an equivalent reaction ratio of CA vs DMAEMA in the hydrogel. Subsequent analysis showed excellent antifouling properties, low blood cell adhesion, hemocompatibility, and platelet deactivation. Moreover, this hydrogel exhibited pH responsiveness to protein adsorption and was then used to facilitate the immobilization of lipase as an indication of active protein functionalization while still maintaining a low fouling status. In summary, a mixed-charge nonfouling pseudozwitterionic hydrogel could be prepared, and its pH-responsive adsorption holds potential for designing a biocompatible tissue engineering matrix or membrane enzyme reactors

Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells

Photocaging provides a method to spatially and temporally control biological function and gene expression with high resolution. Proteins can be photochemically controlled through the site-specific installation of caging groups on amino acid side chains that are essential for protein function. The photocaging of a synthetic gene network using unnatural amino acid mutagenesis in mammalian cells was demonstrated with an engineered bacteriophage RNA polymerase. A caged T7 RNA polymerase was expressed in cells with an expanded genetic code and used in the photochemical activation of genes under control of an orthogonal T7 promoter, demonstrating tight spatial and temporal control. The synthetic gene expression system was validated with two reporter genes (luciferase and EGFP) and applied to the light-triggered transcription of short hairpin RNA constructs for the induction of RNA interference

Genetic incorporation of alkene-lysine analogs 1, 2 and 3 into sfGFP.

<p>(A) SDS-PAGE analysis of purified sfGFP. –AA: no UAA was supplemented; WT: wild-type sfGFP; <b>1</b>, <b>2</b> and <b>3</b>: expression in the presence of the corresponding UAA (1 mM). (B) Protein yields (*wild-type sfGFP yield is 70 mg/L, 100%) and ESI-MS results.</p

Genetic incorporation of alkene-lysine analogs into myoglobin by the wild-type <i>Mb</i>PylRS/PylT_CUA pair.

<p>(A) Structures of alkenyl lysine derivatives bearing an ε-carbamate linkage (<b>1</b>–<b>6</b>), an inverted carbamate <b>7</b>, an amide <b>8</b>, and an urea <b>9</b>. (B) Myoglobin comparative incorporation efficiencies (%) and ESI-MS results.</p

Alkenyl-sfGFP is fluorescently labeled with dansyl-thiol, and bioconjugated to lysozyme to assemble a non-linear protein dimer via the thiol-ene reaction.

<p>(A) sfGFP bearing an alkene functionality reacts photochemically with dansyl-thiol <b>(10)</b> or lysozyme (LYZ). (B) SDS-PAGE analysis demonstrates the labeling of alkenyl-sfGFP with <b>10</b> after 5 min of UV irradiation via thiol-ene ligation (lanes 5 and 6). Fluorescence (top) and Coomassie stain (bottom). (C) SDS-PAGE analysis shows mobility band shifts from 28 kD to 44 kD after samples were UV irradiated for 10 min (lanes 8 and 9), corresponding to the molecular weight of sfGFP-lysozyme conjugate. WT: wild-type sfGFP; <b>1</b> and <b>2</b>: sfGFP carrying the corresponding UAA; LYZ: lysozyme. –UV: samples were not exposed to UV irradiation. +UV: samples were irradiated at 365 nm for 5 or 10 min.</p

Control of Protein Function through Optochemical Translocation

Controlled manipulation of proteins and their function is important in almost all biological disciplines. Here, we demonstrate control of protein activity with light. We present two different applicationslight-triggered transcription and light-triggered protease cleavageboth based on the same concept of protein mislocation, followed by optochemically triggered translocation to an active cellular compartment. In our approach, we genetically encode a photocaged lysine into the nuclear localization signal (NLS) of the transcription factor SATB1. This blocks nuclear import of the protein until illumination induces caging group removal and release of the protein into the nucleus. In the first application, prepending this NLS to the transcription factor FOXO3 allows us to optochemically switch on its transcription activity. The second application uses the developed light-activated NLS to control nuclear import of TEV protease and subsequent cleavage of nuclear proteins containing TEV cleavage sites. The small size of the light-controlled NLS (only 20 amino acids) minimizes impact of its insertion on protein function and promises a general approach to a wide range of optochemical applications. Since the light-activated NLS is genetically encoded and optically triggered, it will prove useful to address a variety of problems requiring spatial and temporal control of protein function, for example, in stem-cell, developmental, and cancer biology

Control of Protein Function through Optochemical Translocation

Controlled manipulation of proteins and their function is important in almost all biological disciplines. Here, we demonstrate control of protein activity with light. We present two different applicationslight-triggered transcription and light-triggered protease cleavageboth based on the same concept of protein mislocation, followed by optochemically triggered translocation to an active cellular compartment. In our approach, we genetically encode a photocaged lysine into the nuclear localization signal (NLS) of the transcription factor SATB1. This blocks nuclear import of the protein until illumination induces caging group removal and release of the protein into the nucleus. In the first application, prepending this NLS to the transcription factor FOXO3 allows us to optochemically switch on its transcription activity. The second application uses the developed light-activated NLS to control nuclear import of TEV protease and subsequent cleavage of nuclear proteins containing TEV cleavage sites. The small size of the light-controlled NLS (only 20 amino acids) minimizes impact of its insertion on protein function and promises a general approach to a wide range of optochemical applications. Since the light-activated NLS is genetically encoded and optically triggered, it will prove useful to address a variety of problems requiring spatial and temporal control of protein function, for example, in stem-cell, developmental, and cancer biology

Control of Protein Function through Optochemical Translocation

Controlled manipulation of proteins and their function is important in almost all biological disciplines. Here, we demonstrate control of protein activity with light. We present two different applicationslight-triggered transcription and light-triggered protease cleavageboth based on the same concept of protein mislocation, followed by optochemically triggered translocation to an active cellular compartment. In our approach, we genetically encode a photocaged lysine into the nuclear localization signal (NLS) of the transcription factor SATB1. This blocks nuclear import of the protein until illumination induces caging group removal and release of the protein into the nucleus. In the first application, prepending this NLS to the transcription factor FOXO3 allows us to optochemically switch on its transcription activity. The second application uses the developed light-activated NLS to control nuclear import of TEV protease and subsequent cleavage of nuclear proteins containing TEV cleavage sites. The small size of the light-controlled NLS (only 20 amino acids) minimizes impact of its insertion on protein function and promises a general approach to a wide range of optochemical applications. Since the light-activated NLS is genetically encoded and optically triggered, it will prove useful to address a variety of problems requiring spatial and temporal control of protein function, for example, in stem-cell, developmental, and cancer biology

Genetically Encoded Optochemical Probes for Simultaneous Fluorescence Reporting and Light Activation of Protein Function with Two-Photon Excitation

The site-specific incorporation of three new coumarin lysine analogues into proteins was achieved in bacterial and mammalian cells using an engineered pyrrolysyl-tRNA synthetase system. The genetically encoded coumarin lysines were successfully applied as fluorescent cellular probes for protein localization and for the optical activation of protein function. As a proof-of-principle, photoregulation of firefly luciferase was achieved in live cells by caging a key lysine residue, and excellent OFF to ON light-switching ratios were observed. Furthermore, two-photon and single-photon optochemical control of EGFP maturation was demonstrated, enabling the use of different, potentially orthogonal excitation wavelengths (365, 405, and 760 nm) for the sequential activation of protein function in live cells. These results demonstrate that coumarin lysines are a new and valuable class of optical probes that can be used for the investigation and regulation of protein structure, dynamics, function, and localization in live cells. The small size of coumarin, the site-specific incorporation, the application as both a light-activated caging group and as a fluorescent probe, and the broad range of excitation wavelengths are advantageous over other genetically encoded photocontrol systems and provide a precise and multifunctional tool for cellular biology