Templated Synthesis of Copper Nanoclusters with a Hybrid Lysozyme-Polymer Material for Enhanced Fluorescence.

Hybrid materials that combine organic polymers and biomacromolecules offer unique opportunities for precisely controlling 3D chemical environments. Although biological or organic templates have been separately used to control the growth of inorganic nanoclusters, hybrid structures represent a relatively unexplored approach to tailoring nanocluster properties. Here, we demonstrate that a molecularly defined lysozyme-polymer resin material acts as a structural scaffold for the synthesis of copper nanoclusters (CuNCs) with well controlled size distributions.

Design of Oscillatory Networks through Post-Translational Control of Network Components.

Many essential functions in biological systems, including cell cycle progression and circadian rhythm regulation, are governed by the periodic behaviors of specific molecules. These periodic behaviors arise from the precise arrangement of components in biomolecular networks that generate oscillatory output signals. The dynamic properties of individual components of these networks, such as maturation delays and degradation rates, often play a key role in determining the network's oscillatory behavior.

A Computational Modeling Approach for the Design of Genetic Control Systems that Respond to Transcriptional Activity.

Recent progress in synthetic biology has enabled the design of complex genetic circuits that interface with innate cellular functions, such as gene transcription, and control user-defined outputs. Implementing these genetic networks in mammalian cells, however, is a cumbersome process that requires several steps of optimization and benefits from the use of predictive modeling. Combining deterministic mathematical models with software-based numerical computing platforms allows researchers to quickly design, evaluate, and optimize multiple circuit topologies to establish experimental constraints that generate the desired control systems.

Boronic Acid Resin for Selective Immobilization of Canonically Encoded Proteins.

The use of transition-metal-mediated boronic acid chemistry presents a novel method of protein immobilization on a solid support. This is a one-step method that site-selectively immobilizes pyroglutamate-histidine (pGH)-tagged proteins. Herein, we describe the synthesis of alkenylboronic acid-functionalized poly(ethylene glycol) acrylamide (PEGA) resin and its subsequent reactions with pGH-tagged proteins to produce covalent linkages.

Cell factory engineering: Challenges and opportunities for synthetic biology applications.

The production of high-quality recombinant proteins is critical to maintaining a continuous supply of biopharmaceuticals, such as therapeutic antibodies. Engineering mammalian cell factories presents a number of limitations typically associated with the proteotoxic stress induced upon aberrant accumulation of off-pathway protein folding intermediates, which eventually culminate in the induction of apoptosis. In this review, we will discuss advances in cell engineering and their applications at different hierarchical levels of control of the expression of recombinant proteins, from transcription and translational to posttranslational modifications and subcellular trafficking.

Emerging technologies for genetic control systems in cellular therapies.

Progress in synthetic biology has enabled the construction of designer cells that sense biological inputs, and, in response, activate user-defined biomolecular programs. Such engineered cells provide unique opportunities for treating a wide variety of diseases. Current strategies mostly rely on cell-surface receptor systems engineered to convert binding interactions into activation of a transcriptional program.

A Platform Technology for Monitoring the Unfolded Protein Response.

The unfolded protein response (UPR) is a complex signal transduction pathway that remodels gene expression in response to proteotoxic stress in the endoplasmic reticulum (ER) and is linked to the development of a range of diseases, including Alzheimer's disease, diabetes, and several types of cancer. UPR induction is typically monitored by measuring the expression level of UPR marker genes. Most tools for quantifying gene expression, including DNA microarrays and quantitative PCR with reverse transcription (RT-PCR), produce snapshots of the cell transcriptome, but are not ideal for measurements requiring temporal resolution of gene expression dynamics.

A platform for post-translational spatiotemporal control of cellular proteins.

Mammalian cells process information through coordinated spatiotemporal regulation of proteins. Engineering cellular networks thus relies on efficient tools for regulating protein levels in specific subcellular compartments. To address the need to manipulate the extent and dynamics of protein localization, we developed a platform technology for the target-specific control of protein destination.

Open questions: how do engineered nanomaterials affect our cells?

Our cells have evolutionarily conserved mechanisms that battle foreign and toxic materials to maintain cellular homeostasis and viability. How do these cellular machineries respond to engineered nanomaterials?

The importance and future of biochemical engineering.

Today's Biochemical Engineer may contribute to advances in a wide range of technical areas. The recent Biochemical and Molecular Engineering XXI conference focused on "The Next Generation of Biochemical and Molecular Engineering: The role of emerging technologies in tomorrow's products and processes". On the basis of topical discussions at this conference, this perspective synthesizes one vision on where investment in research areas is needed for biotechnology to continue contributing to some of the world's grand challenges.

A gene signal amplifier platform for monitoring the unfolded protein response.

Gene expression in mammalian cells results from coordinated protein-driven processes guided by diverse mechanisms of regulation, including protein-protein interactions, protein localization, DNA modifications and chromatin rearrangement. Regulation of gene expression is particularly important in stress-response pathways. To address the need to monitor chromosomal gene expression generating a readily detectable signal output that recapitulates gene expression dynamics, we developed a gene signal amplifier platform that links transcriptional and post-translational regulation of a fluorescent output to the expression of a chromosomal target gene.

Hysteretic Genetic Circuit for Detection of Proteasomal Degradation in Mammalian Cells.

Synthetic hysteretic mammalian gene circuits generating sustained cellular responses to transient perturbations provide important tools to investigate complex cellular behaviors and reprogram cells for a variety of applications, ranging from protein production to cell fate decisions. The design rules of synthetic gene circuits with controlled hysteretic behaviors, however, remain uncharacterized. To identify the criteria for achieving predictable control of hysteresis, we built a genetic circuit for detection of proteasomal degradation (Hys-Deg).

Aggregation Behavior of Nanoparticle-Peptide Systems Affects Autophagy.

The aggregation of nanoparticle colloidal dispersions in complex biological environments changes the nanoparticle properties, such as size and surface area, thus affecting the interaction of nanoparticles at the interface with cellular components and systems. We investigated the effect of nanoparticle aggregation on autophagy, the main catabolic pathway that mediates degradation of nanosized materials and that is activated in response to internalization of foreign nanosized materials. We used carboxylated polystyrene nanoparticles (100 nm) and altered the nanoparticle aggregation behavior through addition of a multidomain peptide, thus generating a set of nanoparticle-peptide mixtures with variable aggregation properties.

Input-dependent post-translational control of the reporter output enhances dynamic resolution of mammalian signaling systems.

Mammalian cells rely on complex and highly dynamic networks that respond to environmental stimuli and intracellular signals and maintain homeostasis. The use of synthetic orthogonal circuits for detection of dynamic behaviors has been limited by the remarkable stability of conventional reporters. While providing an appealing feature for signal amplification, the long half-life of reporters such as GFP is typically not ideal to measure transient signals and dynamic behaviors.

Overcoming component limitations in synthetic biology through transposon-mediated protein engineering.

Protein fission and fusion can be used to create biomolecules with new structures and functions, including circularly permuted proteins that require post-translational modifications for activity, split protein AND gates that require multiple inputs for activity, and fused domains that function as chemical-dependent protein switches. Herein we describe how transposon mutagenesis can be used for protein design to create libraries of permuted, split, or domain-inserted proteins. When coupled with a functional screen or selection, these approaches can rapidly diversify the topologies and functions of natural proteins and create useful protein components for synthetic biology.

A yeast selection system for the detection of proteasomal activation.

The ubiquitin proteasome system (UPS) is a complex cellular machinery that catalyzes degradation of misfolded or damaged proteins and regulates turnover of native proteins in eukaryotic cells, thus playing a crucial role in maintaining protein homeostasis. The UPS has emerged as a drug target for a diverse range of diseases characterized by accumulation of misfolded or aggregated proteins. While enhancement of UPS activity is widely recognized as a promising strategy to prevent accumulation of aberrant, off-pathway protein conformations and ameliorate the phenotypes of a wide range of protein misfolding diseases, the molecular mechanisms underlying activation of proteasomal degradation are poorly characterized.

CLN8 is an endoplasmic reticulum cargo receptor that regulates lysosome biogenesis.

Organelle biogenesis requires proper transport of proteins from their site of synthesis to their target subcellular compartment. Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and traffic through the Golgi complex before being transferred to the endolysosomal system, but how they are transferred from the ER to the Golgi is unknown. Here, we show that ER-to-Golgi transfer of lysosomal enzymes requires CLN8, an ER-associated membrane protein whose loss of function leads to the lysosomal storage disorder, neuronal ceroid lipofuscinosis 8 (a type of Batten disease).

Autophagic response to cellular exposure to titanium dioxide nanoparticles.

Titanium dioxide is "generally regarded as safe" and titanium dioxide nanoparticles (TiO NPs) are used in a wide variety of consumer products. Cellular exposure to TiO NPs results in complex effects on cell physiology including induction of oxidative stress and impairment of lysosomal function, raising concerns about the impact of TiO NPs on biological systems. We investigated the effects of TiO NPs (15, 50, and 100 nm in diameter) on the lysosome-autophagy system, the main cellular catabolic pathway that mediates degradation of nanomaterials.

A Split Transcriptional Repressor That Links Protein Solubility to an Orthogonal Genetic Circuit.

Monitoring the aggregation of proteins within the cellular environment is key to investigating the molecular mechanisms underlying the formation of off-pathway protein assemblies associated with the development of disease and testing therapeutic strategies to prevent the accumulation of non-native conformations. It remains challenging, however, to couple protein aggregation events underlying the cellular pathogenesis of a disease to genetic circuits and monitor their progression in a quantitative fashion using synthetic biology tools. To link the aggregation propensity of a target protein to the expression of an easily detectable reporter, we investigated the use of a transcriptional AND gate system based on complementation of a split transcription factor.

A Naturally Encoded Dipeptide Handle for Bioorthogonal Chan-Lam Coupling.

Manipulation of biomacromolecules is ideally achieved through unique and bioorthogonal chemical reactions of genetically encoded, naturally occurring functional groups. The toolkit of methods for site-specific conjugation is limited by selectivity concerns and a dearth of naturally occurring functional groups with orthogonal reactivity. We report that pyroglutamate amide N-H bonds exhibit bioorthogonal copper-catalyzed Chan-Lam coupling at pyroglutamate-histidine dipeptide sequences.

Quantitatively Predictable Control of Cellular Protein Levels through Proteasomal Degradation.

Protein function is typically studied and engineered by modulating protein levels within the complex cellular environment. To achieve fast, targeted, and predictable control of cellular protein levels without genetic manipulation of the target, we developed a technology for post-translational depletion based on a bifunctional molecule (NanoDeg) consisting of the antigen-binding fragment from the Camelidae species heavy-chain antibody (nanobody) fused to a degron signal that mediates degradation through the proteasome. We provide proof-of-principle demonstration of targeted degradation using a nanobody against the green fluorescent protein (GFP).

TFEB-mediated activation of the lysosome-autophagy system affects the transduction efficiency of adeno-associated virus 2.

Adeno-associated virus (AAV)-mediated gene transfer is an appealing therapeutic option due to AAV's safety profile. Effective delivery of AAV's genetic cargo to the nucleus, however, requires evasion of host cell barriers, including cellular clearance mechanisms mediated by the lysosome-autophagy system. We used AAV serotype 2 to monitor the autophagic response to cellular internalization of AAV and to characterize the effect of AAV-induced activation of autophagy on transgene expression.

Tolerance of a Knotted Near-Infrared Fluorescent Protein to Random Circular Permutation.

Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFPs to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified 27 circularly permuted iRFPs that display biliverdin-dependent fluorescence in Escherichia coli.