Kersten S. Rabe

Orthogonal Protein Decoration of DNA Origami

Structural DNA nanotechnology 2] and the technique of DNA origami enable the rapid generation of a plethora of complex self-assembled nanostructures. Since DNA molecules themselves display limited chemical, optical, and electronic functionality, it is of utmost importance to devise methods to decorate DNA scaffolds with functional moieties to realize applications in sensing, catalysis, and device fabrication. Protein functionalization is particulary desirable because it allows exploitation of an almost unlimited variety of functional elements which nature has evolved over billions of years. The delicate architecture of proteins has resulted in no generally applicable method being currently available to selectively couple these components on DNA scaffolds, and thus approaches used so far are based on reversible antibody– antigen interactions, 9] aptamer binding, 11] nucleic acid hybridization of DNA-tagged proteins, 13] or predominantly biotin–streptavidin (STV) interactions. We demonstrate here that DNA nanostructures can be site-specifically decorated with several different proteins by using coupling systems orthogonal to the biotin–STV system. In particular, benzylguanine (BG) and chlorohexane (CH) groups incorporated in DNA origami have been used as suicide ligands for the site-specific coupling of fusion proteins containing the self-labeling protein tags O-alkylguanine-DNA-alkyltransferase (hAGT), which is often referred to as “Snap-tag”, or haloalkane dehalogenase, which is also known as “HaloTag”. By using various model proteins we demonstrate the general applicability of this approach for the generation of DNA superstructures that are selectively decorated with multiple different proteins. To realize orthogonal protein immobilization on DNA origami using self-ligating protein tags, we chose the Snap-tag, developed by Johnsson and co-workers, and the commercially available HaloTag system. The respective smallmolecule suicide tags (O-benzylguanine (BG) and 5-chlorohexane (CH)) for both self-labeling protein tags are readily available as amino-reactive N-hydroxysuccinimide (NHS) derivatives (BG-NHS and CH-NHS; Figure 1a). Complete derivatization of alkylamino-modified oligonucleotides was achieved by coupling with 30 molar equivalents of BG-NHS or CH-NHS, as indicated by electrophoretic analysis (Figure 1b). To gain access to fusion proteins bearing the complementary Snapand Halo-protein tags, we constructed expression plasmids by genetic fusion of the genes encoding the protein of interest (POI) and Snap-tag or HaloTag (see the Supporting Information). As model POIs we chose the fluorescent proteins enhanced yellow fluorescent protein (EYFP) and mKate, the enzymes cytochrome C peroxidase (CCP) and esterase 2 from Alicyclobacillus acidocaldarius thermos (EST2), to which the self-labeling tags were fused at the C terminus (POI-Snap or POI-Halo, respectively). In addition, the bispecific Halo-Snap fusion protein “covalin”, a chimera which specifically reacts with both BG and CH, as well as monovalent STV (mSTV), were used in this study. The fusion proteins were overexpressed and purified by conventional procedures (see the Supporting Information). The coupling of BGand CHmodified oligonucleotides to the protein was analyzed by using covalin as the initial model to simplify the electrophoretic characterization. It is shown in Figure 1c that both BGand CH-modified single-stranded DNA (ssDNA) oligonucleotides couple effectively to generate the corresponding DNA–covalin conjugates in nearly quantitative yields. DNA coupling of the aforementioned POI fusions, namely mKateSnap, EST2-SNAP, mKate-Halo, CCP-Halo, and EYFP-Halo occurred in a highly specific manner (Figure 1d), and neither Snap or Halo nor mSTV revealed cross-reactivity for the orthogonal-tagged DNA oligomers. We then used SARSE software to aid in the design of face-shaped DNA origami to demonstrate the selective immobilization of protein on DNA nanostructures. Correct folding of M13mp18 ssDNA through the use of 236 staple strands was analyzed by atomic force microscopy (AFM; details of the sequence design as well as experimental procedures are reported in the Supporting Information). Figure 2a illustrates that the face-shaped DNA origami was obtained in high purity, and high-resolution AFM clearly revealed the proposed ears, neck, and seam features of this structure. As an initial test for protein decoration, we selected 23 staple strands, which were biotinylated to create eyes (2 6 [*] Dr. B. Sacc , Dipl.-Chem. R. Meyer, Dipl.-Biotechnol. M. Erkelenz, M. Sc. K. Kiko, A. Arndt, Dr. H. Schroeder, Dr. K. S. Rabe, Prof. C. M. Niemeyer Technische Universit t Dortmund, Fakult t Chemie Biologisch-Chemische Mikrostrukturtechnik Otto-Hahn Strasse 6, 44227 Dortmund (Germany) Fax: (+ 49)231-755-7082 E-mail: christof.niemeyer@tu-dortmund.de [] These authors contributed equally to this work.

Isobutanol production at elevated temperatures in thermophilic Geobacillus thermoglucosidasius

The potential advantages of biological production of chemicals or fuels from biomass at high temperatures include reduced enzyme loading for cellulose degradation, decreased chance of contamination, and lower product separation cost. In general, high temperature production of compounds that are not native to the thermophilic hosts is limited by enzyme stability and the lack of suitable expression systems. Further complications can arise when the pathway includes a volatile intermediate. Here we report the engineering of Geobacillus thermoglucosidasius to produce isobutanol at 50°C. We prospected various enzymes in the isobutanol synthesis pathway and characterized their thermostabilities. We also constructed an expression system based on the lactate dehydrogenase promoter from Geobacillus thermodenitrificans. With the best enzyme combination and the expression system, 3.3g/l of isobutanol was produced from glucose and 0.6g/l of isobutanol from cellobiose in G. thermoglucosidasius within 48h at 50°C. This is the first demonstration of isobutanol production in recombinant bacteria at an elevated temperature.

High‐Throughput Screening for Terpene‐Synthase‐Cyclization Activity and Directed Evolution of a Terpene Synthase

The development of high-throughput assays can be extremely challenging, yet is essential for many applications in drug discovery and enzyme engineering.

Characterization of the peroxidase activity of CYP119, a thermostable P450 from Sulfolobus acidocaldarius.

We report the cloning, expression, and purification of CYP119, a thermostable enzyme previously thought to derive from Sulfolobus solfataricus. Sequence analysis suggested that, in contrast to the conclusions of earlier studies, the enzyme stems from the closely related Sulfolobus acidocaldarius, and we were indeed able to clone the gene from the genomic DNA of this organism. For the first time, we report here on the peroxidase activity of this enzyme and the optimization of the associated reaction parameters. The optimized reaction conditions were then applied to the biocatalytic epoxidation of styrene. The values obtained for kcat (78.2±20.6 min−1) and KM (9.2±4.3 mM) indicated an approximately 100‐fold increased catalytic activity over previously reported results.

Engineering and assaying of cytochrome P450 biocatalysts.

Cytochrome P450s constitute a highly fascinating superfamily of enzymes which catalyze a broad range of reactions. They are essential for drug metabolism and promise industrial applications in biotechnology and biosensing. The constant search for cytochrome P450 enzymes with enhanced catalytic performances has generated a large body of research. This review will concentrate on two key aspects related to the identification and improvement of cytochrome P450 biocatalysts, namely the engineering and assaying of these enzymes. To this end, recent advances in cytochrome P450 development are reported and commonly used screening methods are surveyed.

The Ternary Complex of Cytochrome f and Cytochrome c: Identification of a Second Binding Site and Competition for Plastocyanin Binding

The complex of yeast cytochrome c and cytochrome f from the cyanobacterium Phormidium laminosum was investigated by NMR spectroscopy. Chemical shift perturbation analysis reveals that residues around the haem edge of cytochrome c are involved in the complex interface. Binding curves derived from an NMR spectroscopy titration at 10 mM ionic strength indicate that there are two sites for cytochrome c with binding constants of approximately 2×104 M−1 and 4×103 M−1. A protein docking simulation with NMR‐derived constraints identifies two sites, at the front (Site I) and back faces (Site II) of the haem region of cytochrome f. Site I is homologous to the binding site previously determined for the natural cytochrome f partner plastocyanin. Site II may represent the binding site for the Rieske protein in the cytochrome bf complex. Cytochrome c and plastocyanin are shown to compete for binding at Site I. The competition appears to involve electrostatic screening rather than simple steric occlusion of the binding site.

Photocatalytic Activity of Protein‐Conjugated CdS Nanoparticles

Colloidal CdS nanoparticles are conjugated with a variety of proteins, including enhanced yellow fluorescent protein, tobacco etch virus protease (TEV), lysozyme, and bacterial cytochrome P450 CYP152A1, and the photochemical properties of the resulting conjugates are analyzed by EPR spectroscopy and hydroxyl radical-specific fluorimetric assay. While irradiation of bare CdS colloids leads to photogeneration of hydroxyl and superoxide radicals, it is surprisingly observed that coating of the CdS particles with proteins effectively suppresses the production of these radical species and instead leads to increased formation of a long-lived reactive oxygen species, most likely H(2)O(2). A mechanism for the observed results is suggested. The empirical results are capitalized on for the assembly of a CdS-TEV nanohybrid, which shows significantly higher performance as a photocatalytic mediator for fatty acid hydroxylation by CYP152A1 than bare CdS nanoparticles.

The esterase from Alicyclobacillus acidocaldarius as a reporter enzyme and affinity tag for protein biosynthesis.

Esterase from thermophilic bacteria Alicyclobacillus acidocaldarius can be produced up to 200 μg/ml by coupled in vitro transcription/translation system derived from Escherichia coli. The synthesized thermostable enzyme can be determined by photometrical and fluorescent assays at least up to 10−8 M concentration or by activity staining in the polyacrylamide gels. Enhanced green fluorescence protein‐esterase fusion protein was bound to a matrix with immobilized esterase inhibitor and purified by affinity chromatography. Thus, the esterase is suited as a reporter enzyme to monitor the expression of polypeptides coupled to its N‐terminus and simultaneously, as a cleavable tag for polypeptide purification.

Cascades in Compartments: En route to Machine-Assisted Biotechnology

Biological compartmentalization is a fundamental principle of life that allows cells to metabolize, propagate, or communicate with their environment. Much research is devoted to understanding this basic principle and to harness biomimetic compartments and catalytic cascades as tools for technological processes. This Review summarizes the current state-of-the-art of these developments, with a special emphasis on length scales, mass transport phenomena, and molecular scaffolding approaches, ranging from small cross-linkers over proteins and nucleic acids to colloids and patterned surfaces. We conclude that the future exploration and exploitation of these complex systems will largely benefit from technical solutions for the integrated, machine-assisted development and maintenance of a next generation of biotechnological processes. These goals should be achievable by implementing microfluidics, robotics, and added manufacturing techniques supplemented by theoretical simulations as well as computer-aided process modeling based on big data obtained from multiscale experimental analyses.

Multifunctional Silica Nanoparticles for Covalent Immobilization of Highly Sensitive Proteins.

A convenient reverse micellar one-pot reaction yields multifunctional silica nanoparticles, which can be tailored to effectively suppress non-specific adsorption and, at the same time, enable efficient specific covalent immobilization of proteins. Using two highly sensitive proteins, it is demonstrated that the new particles provide a suitable microenvironment to maintain the proteins activity.