Cell-free technologies for biopharmaceutical research and production.

Cell-free protein synthesis (CFPS) technologies have grown from lab-scale research tools to biopharmaceutical production at the Good Manufacturing Practice manufacturing scale. Multiple human clinical trials are in progress with CFPS-based products. In addition, applications of CFPS in research have continued to expand over the years and play an important role in biopharmaceutical product discovery and development.

Expanding biological applications using cell-free metabolic engineering: An overview.

Expanding the concept of cell-free biology, implemented both with purified components and crude extracts, is continuing to deepen our appreciation of biological fundamentals while enlarging the range of applications. We are no longer intimidated by the complexity of crude extracts and complicated reaction systems with hundreds of active components, and, instead, coordinately activate and inactivate metabolic processes to focus and expand the capabilities of natural biological processes. This, in turn, dramatically increases the range of benefits offered by new products, both natural and supernatural, that were previously infeasible and/or unimaginable.

System analysis and improved [FeFe] hydrogenase O tolerance suggest feasibility for photosynthetic H production.

Photosynthetic H production has been a compelling but elusive objective. Here we describe how coordinated bioreactor, metabolic pathway, and protein engineering now suggest feasibility for the sustainable, solar-powered production of a storable fuel to complement our expanding photovoltaic and wind based capacities. The need to contain and harvest the gaseous products provides decisive solar bioreactor design advantages by limiting O exposure to prolific, but O-sensitive H producing enzymes-[FeFe] hydrogenases.

Streamlined circular proximity ligation assay provides high stringency and compatibility with low-affinity antibodies.

Proximity ligation assay (PLA) is a powerful tool for quantitative detection of protein biomarkers in biological fluids and tissues. Here, we present the circular proximity ligation assay (c-PLA), a highly specific protein detection method that outperforms traditional PLA in stringency, ease of use, and compatibility with low-affinity reagents. In c-PLA, two proximity probes bind to an analyte, providing a scaffolding that positions two free oligonucleotides such that they can be ligated into a circular DNA molecule.

Virus-like particles: Next-generation nanoparticles for targeted therapeutic delivery.

Most drug therapies distribute the agents throughout the entire body, even though the drugs are typically only needed at specific tissues. This often limits dosage and causes discomfort and harmful side-effects. Significant research has examined nanoparticles (NPs) for use as targeted delivery vehicles for therapeutic cargo, however, major clinical success has been limited.

High-Throughput Screening of Catalytic H Production.

Hydrogenases, ferredoxins, and ferredoxin-NADP reductases (FNR) are redox proteins that mediate electron metabolism in vivo, and are also potential components for biological H production technologies. A high-throughput H production assay device (H PAD) is presented that enables simultaneous evaluation of 96 individual H production reactions to identify components that improve performance. Using a CCD camera and image analysis software, H PAD senses the chemo-optical response of Pd/WO thin films to the H produced.

Characterization of [FeFe] Hydrogenase O2 Sensitivity Using a New, Physiological Approach.

[FeFe] hydrogenases catalyze rapid H production but are highly O-sensitive. Developing O-tolerant enzymes is needed for sustainable H production technologies, but the lack of a quantitative and predictive assay for O tolerance has impeded progress. We describe a new approach to provide quantitative assessment of O sensitivity by using an assay employing ferredoxin NADP reductase (FNR) to transfer electrons from NADPH to hydrogenase via ferredoxins (Fd).

The Radical SAM Enzyme HydG Requires Cysteine and a Dangler Iron for Generating an Organometallic Precursor to the [FeFe]-Hydrogenase H-Cluster.

Three maturase enzymes-HydE, HydF, and HydG-synthesize and insert the organometallic component of the [FeFe]-hydrogenase active site (the H-cluster). HydG generates the first organometallic intermediates in this process, ultimately producing an [Fe(CO)2(CN)] complex. A limitation in understanding the mechanism by which this complex forms has been uncertainty regarding the precise metallocluster composition of HydG that comprises active enzyme.

Functional properties of flagellin as a stimulator of innate immunity.

We report the development of a well-defined flagellin-based nanoparticle stimulator and also provide a new mechanism of action model explaining how flagellin-triggered innate immunity has evolved to favor localized rather than potentially debilitating systemic immune stimulation. Cell-free protein synthesis (CFPS) was used to facilitate mutational analysis and precisely orientated display of flagellin on Hepatitis B core (HBc) protein virus-like particles (VLPs). The need for product stability and an understanding of mechanism of action motivated investigations indicating that the D0 domain of flagellin is sensitive to amino acid sequence independent hydrolysis - apparently due to the need for structural flexibility during natural flagellin polymerization.

Biosynthesis of the [FeFe] Hydrogenase H Cluster: A Central Role for the Radical SAM Enzyme HydG.

Hydrogenase enzymes catalyze the rapid and reversible interconversion of H2 with protons and electrons. The active site of the [FeFe] hydrogenase is the H cluster, which consists of a [4Fe-4S]H subcluster linked to an organometallic [2Fe]H subcluster. Understanding the biosynthesis and catalytic mechanism of this structurally unusual active site will aid in the development of synthetic and biological hydrogenase catalysts for applications in solar fuel generation.

Assessing sequence plasticity of a virus-like nanoparticle by evolution toward a versatile scaffold for vaccines and drug delivery.

Virus-like particles (VLPs) have been extensively explored as nanoparticle vehicles for many applications in biotechnology (e.g., vaccines, drug delivery, imaging agents, biocatalysts).

Cysteine as a ligand platform in the biosynthesis of the FeFe hydrogenase H cluster.

Hydrogenases catalyze the redox interconversion of protons and H2, an important reaction for a number of metabolic processes and for solar fuel production. In FeFe hydrogenases, catalysis occurs at the H cluster, a metallocofactor comprising a [4Fe-4S]H subcluster coupled to a [2Fe]H subcluster bound by CO, CN(-), and azadithiolate ligands. The [2Fe]H subcluster is assembled by the maturases HydE, HydF, and HydG.

Chemical lysis of cyanobacteria.

We have developed a mixture of enzymes and chemicals that completely lyse cyanobacteria. Since the treatment involves only readily-available chemicals and simple proteins that degrade the components of the cyanobacterial cell wall, it can easily be used in high-throughput applications requiring lysis for subsequent intracellular measurements. Our lysis technique consistently enables complete lysis of several different cyanobacterial strains, and we demonstrated that DNA, mRNA, and proteins are preserved in the lysates.

X-ray crystallographic and EPR spectroscopic analysis of HydG, a maturase in [FeFe]-hydrogenase H-cluster assembly.

Hydrogenases use complex metal cofactors to catalyze the reversible formation of hydrogen. In [FeFe]-hydrogenases, the H-cluster cofactor includes a diiron subcluster containing azadithiolate, three CO, and two CN(-) ligands. During the assembly of the H cluster, the radical S-adenosyl methionine (SAM) enzyme HydG lyses the substrate tyrosine to yield the diatomic ligands.

The cyanide ligands of [FeFe] hydrogenase: pulse EPR studies of (13)C and (15)N-labeled H-cluster.

The two cyanide ligands in the assembled cluster of [FeFe] hydrogenase originate from exogenous l-tyrosine. Using selectively labeled tyrosine substrates, the cyanides were isotopically labeled via a recently developed in vitro maturation procedure allowing advanced electron paramagnetic resonance techniques to probe the electronic structure of the catalytic core of the enzyme. The ratio of the isotropic (13)C hyperfine interactions for the two CN(-) ligands-a reporter of spin density on their respective coordinating iron ions-collapses from ≈5.

Cell-free synthesis of the H-cluster: a model for the in vitro assembly of metalloprotein metal centers.

Many organometallic cofactors are highly complex and require multiple accessory proteins for both their assembly and transfer to a target protein. A cell-free system in which the biosynthetic pathway for a prosthetic group has been fully or even partially reconstructed enables investigations of the reaction sequence as well as the cofactor itself. As a model for the in vitro assembly of protein-bound metal centers, we describe a procedure for the cell-free synthesis of the H-cluster in the context of producing purified and active [FeFe] hydrogenase samples for spectroscopic studies.

The HydG enzyme generates an Fe(CO)2(CN) synthon in assembly of the FeFe hydrogenase H-cluster.

Three iron-sulfur proteins--HydE, HydF, and HydG--play a key role in the synthesis of the [2Fe](H) component of the catalytic H-cluster of FeFe hydrogenase. The radical S-adenosyl-L-methionine enzyme HydG lyses free tyrosine to produce p-cresol and the CO and CN(-) ligands of the [2Fe](H) cluster. Here, we applied stopped-flow Fourier transform infrared and electron-nuclear double resonance spectroscopies to probe the formation of HydG-bound Fe-containing species bearing CO and CN(-) ligands with spectroscopic signatures that evolve on the 1- to 1000-second time scale.

Production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines.

The rapid dissemination of the 2009 pandemic H1N1 influenza virus emphasizes the need for universal influenza vaccines that would broadly protect against multiple mutated strains. Recent efforts have focused on the highly conserved hemagglutinin (HA) stem domain, which must undergo a significant conformational change for effective viral infection. Although the production of isolated domains of multimeric ectodomain proteins has proven difficult, we report a method to rapidly produce the properly folded HA stem domain protein from influenza virus A/California/05/2009 (H1N1) by using Escherichia coli-based cell-free protein synthesis and a simple refolding protocol.

Direct polymerization of proteins.

We report the synthesis of active polymers of superfolder green fluorescent protein (sfGFP) in one step using Click chemistry. Up to six copies of the non-natural amino acids (nnAAs) p-azido-l-phenylalanine (pAzF) or p-propargyloxy-l-phenylalanine (pPaF) were site-specifically inserted into sfGFP by cell-free protein synthesis (CFPS). sfGFP containing two or three copies of these nnAAs were coupled by copper-catalyzed azide-alkyne cycloaddition to synthesize linear or branched protein polymers, respectively.

A radical intermediate in tyrosine scission to the CO and CN- ligands of FeFe hydrogenase.

The radical S-adenosylmethionine (SAM) enzyme HydG lyses free l-tyrosine to produce CO and CN(-) for the assembly of the catalytic H cluster of FeFe hydrogenase. We used electron paramagnetic resonance spectroscopy to detect and characterize HydG reaction intermediates generated with a set of (2)H, (13)C, and (15)N nuclear spin-labeled tyrosine substrates. We propose a detailed reaction mechanism in which the radical SAM reaction, initiated at an N-terminal 4Fe-4S cluster, generates a tyrosine radical bound to a C-terminal 4Fe-4S cluster.

Cell-free co-production of an orthogonal transfer RNA activates efficient site-specific non-natural amino acid incorporation.

We describe a new cell-free protein synthesis (CFPS) method for site-specific incorporation of non-natural amino acids (nnAAs) into proteins in which the orthogonal tRNA (o-tRNA) and the modified protein (i.e. the protein containing the nnAA) are produced simultaneously.

Escherichia coli-based cell free production of flagellin and ordered flagellin display on virus-like particles.

Bacterial flagellin has been explored as a potential vaccine adjuvant for enhancing immune responses. In this article, we describe Escherichia coli-based cell-free protein synthesis (CFPS) as a method to rapidly produce soluble phase 1 flagellin (FliC) protein from Salmonella typhimurium. The yield was about 300 µg/mL and the product had much higher affinity for the TLR5 receptor (EC50 = 2.

Pluripotency transcription factor Sox2 is strongly adsorbed by heparin but requires a protein transduction domain for cell internalization.

The binding of protein transduction domain (PTD)-conjugated proteins to heparan sulfate is an important step in cellular internalization of macromolecules. Here, we studied the pluripotency transcription factor Sox2, with or without the nonaarginine (R9) PTD. Unexpectedly, we observed that Sox2 is strongly adsorbed by heparin and by the fibroblasts without the R9 PTD.

Using E. coli-based cell-free protein synthesis to evaluate the kinetic performance of an orthogonal tRNA and aminoacyl-tRNA synthetase pair.

Even though the orthogonal tRNA and aminoacyl-tRNA synthetase pairs derived from the archaeon Methanocaldococcus jannaschii have been used for many years for site-specific incorporation of non-natural amino acids (nnAAs) in Escherichia coli, their kinetic parameters have not been evaluated. Here we use a cell-free protein synthesis (CFPS) system to control the concentrations of the orthogonal components in order to evaluate their performance while supporting synthesis of modified proteins (i.e.

Nuclear resonance vibrational spectroscopy and electron paramagnetic resonance spectroscopy of 57Fe-enriched [FeFe] hydrogenase indicate stepwise assembly of the H-cluster.

The [FeFe] hydrogenase from Clostridium pasteurianum (CpI) harbors four Fe-S clusters that facilitate the transfer of an electron to the H-cluster, a ligand-coordinated six-iron prosthetic group that catalyzes the redox interconversion of protons and H(2). Here, we have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the iron centers in CpI, and we compare our data to that for a [4Fe-4S] ferredoxin as well as a model complex resembling the [2Fe](H) catalytic domain of the H-cluster. To enrich the hydrogenase with (57)Fe nuclei, we used cell-free methods to post-translationally mature the enzyme.