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Joonbaek Jang received his B.S. from Korea University in 2016. He then moved to the University of Pennsylvania where he obtained his M.S. working with Prof. Raymond J. Gorte. During his master’s degree, Joonbaek used atomic layer deposition to modify metal oxide catalysts for the improvement of industrial catalytic processes. In 2018, he joined the Morales-Guio Lab in the Department of Chemical and Biomolecular Engineering at UCLA. Joonbaek is currently working toward the understanding of electrode/electrolyte interfaces and mass transport effects in electrocatalytic processes for sustainable energy applications. Kangze Shen received his B.S. in Materials Chemistry from University of Science and Technology of China in 2018. His undergraduate research with Prof. Yitai Qian focused on the development of high-performance electrode materials for rechargeable Li-ion and Na-ion batteries. He then joined the Morales-Guio Lab in the Department of Chemical and Biomolecular Engineering at UCLA where he is working toward the understanding of electrocatalytic processes and electrochemical systems engineering in sustainable energy applications. Carlos G. Morales-Guio is an Assistant Professor of Chemical and Biomolecular Engineering at UCLA. The Morales-Guio Lab is interested in electrochemical catalysis, particularly with respect to energy and chemical transformations for sustainable energy applications. The lab is currently focused on understanding fundamentals of mass and charge transport coupled to electrochemical transformations that will guide the scale up of electrocatalytic reactors. Carlos received his B. Eng. degree from Osaka University in 2011 and his M.S. and Ph.D. in Chemistry and Chemical Engineering from EPFL in 2013 and 2016, respectively. Most recently, he was a postdoctoral research fellow at Stanford University before joining UCLA in 2018.

One of the central themes of biomolecular engineering is the challenge of exploiting the properties of biological materials. Part of this challenge has been uncovering and harnessing properties of biological components that only emerge following their ordered self-assembly. One biomolecular building block that has received significant interest in the past decade is the M13 bacteriophage. There have been a number of recent attempts to trigger the ordered assembly of M13 bacteriophage into multivirion structures, relying on the innate tendency of M13 to form liquid crystals at high concentrations. These, in general, yield planar two-dimensional materials. Presented here is the production of multivirion assemblies of M13 bacteriophage via the chemical modification of its surface by the covalent attachment of the xanthene-based dye tetramethylrhodamine (TMR) isothiocyanate (TRITC). We show that TMR induces the formation of three-dimensional aster-like assemblies of M13 by providing "adhesive" action between bacteriophage particles through the formation of H-aggregates (face-to-face stacking of dye molecules). We also show that the H-aggregation of TMR is greatly enhanced by covalent attachment to M13 and is enhanced further still upon the ordered self-assembly of M13, leading to the suggestion that M13 could be used to promote the self-assembly of dyes that form J-aggregates, a desirable arrangement of fluorescent dye, which has interesting optical properties and potential applications in the fields of medicine and light harvesting technology.

The increased availability of user-friendly and accessible computational tools for biomolecular modeling would expand the reach and application of biomolecular engineering and design. For protein modeling, one key challenge is to reduce the complexities of 3D protein folds to sets of parametric equations that nonetheless capture the salient features of these structures accurately. At present, this is possible for a subset of proteins, namely, repeat proteins. The α-helical coiled coil provides one such example, which represents ≈ 3-5% of all known protein-encoding regions of DNA. Coiled coils are bundles of α helices that can be described by a small set of structural parameters. Here we describe how this parametric description can be implemented in an easy-to-use web application, called CCBuilder 2.0, for modeling and optimizing both α-helical coiled coils and polyproline-based collagen triple helices. This has many applications from providing models to aid molecular replacement for X-ray crystallography, in silico model building and engineering of natural and designed protein assemblies, and through to the creation of completely de novo "dark matter" protein structures. CCBuilder 2.0 is available as a web-based application, the code for which is open-source and can be downloaded freely. http://coiledcoils.chm.bris.ac.uk/ccbuilder2. Lay summary We have created CCBuilder 2.0, an easy to use web-based application that can model structures for a whole class of proteins, the α-helical coiled coil, which is estimated to account for 3-5% of all proteins in nature. CCBuilder 2.0 will be of use to a large number of protein scientists engaged in fundamental studies, such as protein structure determination, through to more-applied research including designing and engineering novel proteins that have potential applications in biotechnology.

Immobilized antibody systems are the key to develop efficient diagnostics and separations tools. In the last decade, developments in the field of biomolecular engineering and crosslinker chemistry have greatly influenced the development of this field. With all these new approaches at our disposal, several new immobilization methods have been created to address the main challenges associated with immobilized antibodies. Few of these challenges that we have discussed in this review are mainly associated to the site-specific immobilization, appropriate orientation, and activity retention. We have discussed the effect of antibody immobilization approaches on the parameters on the performance of an immunoassay.

Engineering materials using supramolecular principles enables generalizable and modular platforms that have tunable chemical, mechanical, and biological properties. Applying this bottom‐up, molecular engineering‐based approach to therapeutic design affords unmatched control of emergent properties and functionalities. In preparing responsive materials for biomedical applications, the dynamic character of typical supramolecular interactions facilitates systems that can more rapidly sense and respond to specific stimuli through a fundamental change in material properties or characteristics, as compared to cases where covalent bonds must be overcome. Several supramolecular motifs have been evaluated toward the preparation of “smart” materials capable of sensing and responding to stimuli. Triggers of interest in designing materials for therapeutic use include applied external fields, environmental changes, biological actuators, applied mechanical loading, and modulation of relative binding affinities. In addition, multistimuli‐responsive routes can be realized that capture combinations of triggers for increased functionality. In sum, supramolecular engineering offers a highly functional strategy to prepare responsive materials. Future development and refinement of these approaches will improve precision in material formation and responsiveness, seek dynamic reciprocity in interactions with living biological systems, and improve spatiotemporal sensing of disease for better therapeutic deployment.

Halogenation is widespread in Nature, as witnessed by the several thousands of naturally occurring halogenated compounds. This review seeks to provide an overview on the pathways and multiple roles of halogenation in Nature, as a source of inspiration to chemists. Remarkably, several halogenation pathways were recently found in humans, sparking an increasing interest into halogens as biomolecular recognition sites. A description of the molecular mechanisms underlining the role of halogenation in the biomolecular context is provided, highlighting how the strategic use of halogen-driven interactions can be exploited to tune the properties of both small compounds and macromolecules. The complexity, versatility and bio-orthogonality of halogen-driven interactions are indeed key figures of merit in promoting novel opportunities for halogenation in a range of applications, spanning from drug design to self-assembly and biomolecular engineering. Specifically, the most relevant and impactful recent contributions in the field of peptide and protein science are presented and critically discussed, with a particular emphasis on the existing opportunities to control their conformational and self-assembly properties towards novel therapeutics and functional materials.

Collagen-based biomedical materials have developed into important, clinically effective materials used in a range of devices that have gained wide acceptance. These devices come with collagen in various formats, including those based on stabilized natural tissues, those that are based on extracted and purified collagens, and designed composite, biosynthetic materials. Further knowledge on the structure and function of collagens has led to on-going developments and improvements. Among these developments has been the production of recombinant collagen materials that are well defined and are disease free. Most recently, a group of bacterial, non-animal collagens has emerged that may provide an excellent, novel source of collagen for use in biomaterials and other applications. These newer collagens are discussed in detail. They can be modified to direct their function, and they can be fabricated into various formats, including films and sponges, while solutions can also be adapted for use in surface coating technologies.

In nature, assembled protein structures offer the most complex functional structures. The understanding of the mechanisms ruling protein-protein interactions opens the door to manipulate protein assemblies in a rational way. Proteins are versatile scaffolds with great potential as tools in nanotechnology and biomedicine because of their chemical, structural, and functional versatility. Currently, bottom-up self-assembly based on biomolecular interactions of small and well-defined components, is an attractive approach to biomolecular engineering and biomaterial design. Specifically, repeat proteins are simplified systems for this purpose. In this work, we provide an overview of fundamental concepts of the design of new protein interfaces. We describe an experimental approach to form higher order architectures by a bottom-up assembly of repeated building blocks. For this purpose, we use designed consensus tetratricopeptide repeat proteins (CTPRs). CTPR arrays contain multiple identical repeats that interact through a single inter-repeat interface to form elongated superhelices. Introducing a novel interface along the CTPR superhelix allows two CTPR molecules to assemble into protein nanotubes. We apply three approaches to form protein nanotubes: electrostatic interactions, hydrophobic interactions, and π-π interactions. We isolate and characterize the stability and shape of the formed dimers and analyze the nanotube formation considering the energy of the interaction and the structure in the three different models. These studies provide insights into the design of novel protein interfaces for the control of the assembly into more complex structures, which will open the door to the rational design of nanostructures and ordered materials for many potential applications in nanotechnology.

Virus-like particles (VLPs) are repetitive organizations of viral proteins assembled in an appropriate physicochemical environment. VLPs can stimulate both innate and adaptive immune responses, due to their particulate structure enabling uptake by antigen presenting cells. These characteristics have led to successful development of VLP-vaccine products, and will ensure their vast potential in years to come. Future success of VLP therapeutic products will be determined by advances in their bioengineering, and also by the development of tools to design for their stability, function and application. This review focuses on approaches for VLP assembly in controlled chemical environments in vivo and in vitro, and the application of computational tools for improved chemical sequence design, and fundamental understanding of assembly.

Biomolecular engineering can be used to purposefully manipulate biomolecules, such as peptides, proteins, nucleic acids and lipids, within the framework of the relations among their structures, functions and properties, as well as their applicability to such areas as developing novel biomaterials, biosensing, bioimaging, and clinical diagnostics and therapeutics. Nanotechnology can also be used to design and tune the sizes, shapes, properties and functionality of nanomaterials. As such, there are considerable overlaps between nanotechnology and biomolecular engineering, in that both are concerned with the structure and behavior of materials on the nanometer scale or smaller. Therefore, in combination with nanotechnology, biomolecular engineering is expected to open up new fields of nanobio/bionanotechnology and to contribute to the development of novel nanobiomaterials, nanobiodevices and nanobiosystems. This review highlights recent studies using engineered biological molecules (e.g., oligonucleotides, peptides, proteins, enzymes, polysaccharides, lipids, biological cofactors and ligands) combined with functional nanomaterials in nanobio/bionanotechnology applications, including therapeutics, diagnostics, biosensing, bioanalysis and biocatalysts. Furthermore, this review focuses on five areas of recent advances in biomolecular engineering: (a) nucleic acid engineering, (b) gene engineering, (c) protein engineering, (d) chemical and enzymatic conjugation technologies, and (e) linker engineering. Precisely engineered nanobiomaterials, nanobiodevices and nanobiosystems are anticipated to emerge as next-generation platforms for bioelectronics, biosensors, biocatalysts, molecular imaging modalities, biological actuators, and biomedical applications.

Azobenzene has been widely used as a photoregulator due to its reversible photoisomerization, large structural change between E and Z isomers, high photoisomerization yield, and high chemical stability. On the other hand, some azobenzene derivatives can be used as universal quenchers for many fluorophores. Nucleic acid is a good candidate to be modified because it is not only the template of gene expression but also widely used for building well-organized nanostructures and nanodevices. Because the size and polarity distribution of the azobenzene molecule is similar to a nucleobase pair, the introduction of azobenzene into nucleic acids has been shown to be an ingenious molecular design for constructing light-switching biosystems or light-driven nanomachines. Here we review recent advances in azobenzene-modified nucleic acids and their applications for artificial regulation of gene expression and enzymatic reactions, construction of photoresponsive nanostructures and nanodevices, molecular beacons, as well as obtaining structural information using the introduced azobenzene as an internal probe. In particular, nucleic acids bearing multiple azobenzenes can be used as a novel artificial nanomaterial with merits of high sequence specificity, regular duplex structure, and high photoregulation efficiency. The combination of functional groups with biomolecules may further advance the development of chemical biotechnology and biomolecular engineering.

Control over the functionality of interfaces through biomolecular engineering is a central tool for nanoscale technology as well as many current applications of biology. In this work we designed fusion proteins that combined the surface adhesion and interfacial activity of a hydrophobin-protein together with the high affinity biotin-binding capability of an avidin-protein. We found that an overall architecture that was based on a circularly permuted version of avidin, dual-chain avidin, and hydrophobin gave a highly functional combination. The protein was produced in the filamentous fungus Trichoderma reesei and was efficiently purified using an aqueous two-phase partitioning procedure. The surface adhesive properties were widely different compared to wild-type avidin. Functional characterization showed that the protein assembled on hydrophobic surfaces as a thin layer even at very low concentrations and efficiently bound a biotinylated compound. The work shows how the challenge of creating a fusion protein with proteins that form multimers can be solved by structural design and how protein self-assembly can be used to efficiently functionalize interfaces.

Folding-based electrochemical sensors utilizing structure-switching aptamers are specific, selective, sensitive, and widely applicable to the detection of a variety of target analytes. The specificity is achieved by the binding properties of an electrode-bound RNA or DNA aptamer biorecognition element. Signaling in this class of sensors arises from changes in electron transfer efficiency upon target-induced changes in the conformation/flexibility of the aptamer probe. These changes can be readily monitored electrochemically. Because of this signaling mechanism, there are several approaches to maximizing the analytical attributes (i.e., sensitivity, limit of detection, and observed binding affinity) of the aptamer sensor. Here, we present a systematic study of several approaches, including electrochemical interrogation parameters and biomolecular engineering of the aptamer sequence, to develop a sensor for the detection of aminoglycoside antibiotics. Specifically, through a combination of optimizing the electrochemical signal and engineering the parent 26-nucleotide RNA aptamer sequence to undergo larger conformation changes, we develop several improved sensors. These sensors exhibit binding affinities ranging from 220 nM to 42 μM, as much as a 100-fold improved limit of detection in comparison to previously reported sensors, and a variety of linear ranges including the therapeutic window for tobramycin. These data demonstrate that rational engineering of the aptamer structure to create large conformation changes upon target binding leads to improved sensor performance. We believe that the sensor design guidelines outlined here represent a general strategy for developing new aptamer folding-based electrochemical sensors.

Si-Shen Feng Author for correspondence: Department of Chemical & Biomolecular Engineering, Department of Bioengineering, & Nanoscience & Nanotechnology Initiative (NUSNNI/NanoCore), National University of Singapore, Block E3, 05–29, 2 Engineering Drive 3, 117576, Singapore Tel.: +65 6576 3835 Fax: +65 6779 1936 chefess@nus.edu.sg Star-shaped block polymers as a molecular biomaterial for nanomedicine development

Nanomedicine is an emerging field that integrates nanotechnology, biomolecular engineering, life sciences and medicine; it is expected to produce major breakthroughs in medical diagnostics and therapeutics. Nano-scale structures and devices are compatible in size with proteins and nucleic acids in living cells. Therefore, the design, characterization and application of nano-scale probes, carriers and machines may provide unprecedented opportunities for achieving a better control of biological processes, and drastic improvements in disease detection, therapy, and prevention. Recent advances in nanomedicine include the development of nanoparticle-based probes for molecular imaging, nano-carriers for drug/gene delivery, multi-functional nanoparticles for theranostics, and molecular machines for biological and medical studies. This article provides an overview of the nanomedicine field, with an emphasis on nanoparticles for imaging and therapy, as well as engineered nucleases for genome editing. The challenges in translating nanomedicine approaches to clinical applications are discussed.

Natural products are complex small molecules produced by an array of organisms and which humans have put to diverse uses, including as pesticides, antibiotics, and anticancer agents. While a wide range has already been uncovered it is clear that there are many more potential biosynthetic pathways for which the products have never been studied. Using the tools of synthetic biology allows us to manipulate these pathways and produce the desired compounds. The genes involved can also be transferred into other organisms which can be more easily manipulated. Recent advances in understanding these complex systems have also informed the targeted manipulation of biosynthetic pathways to produce specific modifications of the final natural product. In the future it may be possible, rather than relying on chemical synthesis, to design and build artificial pathways to synthesize completely new compounds as desired.

Many important biological processes – ranging from simple metabolism to complex cognition – take place deep inside living organisms, yet our ability to study them in this context is very limited. Technologies such as fluorescent proteins and optogenetics enable exquisitely precise imaging and control of cellular function in small, translucent specimens using visible light, but are limited by the poor penetration of such light into larger tissues. In contrast, most non-invasive technologies such as magnetic resonance imaging (MRI) and ultrasound – while based on energy forms that penetrate tissue effectively – lack the needed molecular precision. Our work attempts to bridge this gap by engineering new molecular technologies that connect penetrant energy to specific aspects of cellular function in vivo. In this talk, I will describe molecular reporters for non-invasive imaging using MRI and ultrasound developed by adapting and engineering naturally occurring proteins. These proteins have physical properties, such as paramagnetism or self-assembly into hollow nanostructures, that allow them to be sensitively detected with MRI and ultrasound. By engineering them at the genetic level, we have adapted these natural constructs into noninvasive molecular reporters of biological processes ranging from gene expression to chemical neurotransmission and metabolism. In addition, I will describe recent work on the use of penetrant forms of energy to control cellular function within the body.

New strategies to self-assemble biocompatible materials into nanoscale, drug-loaded packages with improved therapeutic efficacy are needed for nanomedicine. To address this need, we developed artificial recombinant chimeric polypeptides (CPs) that spontaneously self-assemble into sub-100 nm size, near monodisperse nanoparticles upon conjugation of diverse hydrophobic molecules, including chemotherapeutics. These CPs consist of a biodegradable polypeptide that is attached to a short Cys-rich segment. Covalent modification of the Cys residues with a structurally diverse set of hydrophobic small molecules, including chemotherapeutics leads to spontaneous formation of nanoparticles over a range of CP compositions and molecular weights. When used to deliver chemotherapeutics to a murine cancer model, CP nanoparticles have a four-fold higher maximum tolerated dose than free drug, and induce nearly complete tumor regression after a single dose. This simple strategy can promote co-assembly of drugs, imaging agents, and targeting moieties into multifunctional nanomedicines.

Spider silk is an extraordinary material with physical properties comparable to the best scaffolding/structural materials, and as a fiber it can be manipulated with ease into a variety of configurations. Our work here demonstrates that natural spider silk fibers can also be used to organize biological components on and in devices through rapid and simple means. Micron scale spider silk fibers (5-10 μm in diameter) were surface modified with a variety of biological entities engineered with pentaglutamine tags via microbial transglutaminase (mTG). Enzymes, enzyme pathways, antibodies, and fluorescent proteins were all assembled onto spider silk fibers using this biomolecular engineering/biofabrication process. Additionally, arrangement of biofunctionalized fiber should in of itself generate a secondary level of biomolecular organization. Toward this end, as proofs of principle, spatially defined arrangement of biofunctionalized spider silk fiber was shown to generate effects specific to silk position in two cases. In one instance, arrangement perpendicular to a flow produced selective head and neck carcinoma cell capture on silk with antibodies complexed to conjugated protein G. In a second scenario, asymmetric bacterial chemotaxis arose from asymmetric conjugation of enzymes to arranged silk. Overall, the biofabrication processes used here were rapid, required no complex chemistries, were biologically benign, and also the resulting engineered silk microfibers were flexible, readily manipulated and functionally active. Deployed here in microfluidic environments, biofunctional spider silk fiber provides a means to convey complex biological functions over a range of scales, further extending its potential as a biomaterial in biotechnological settings. Biotechnol. Bioeng. 2017;114: 83-95. © 2016 Wiley Periodicals, Inc.

Due to their structural and mechanical properties, 1D helical protein assemblies represent highly attractive design targets for biomolecular engineering and protein design. Here we present a designed, tetrameric protein building block, Zn(8)R(4), which assembles via Zn coordination interactions into a series of kinetically stable, crystalline, helical nanotubes whose widths can be controlled by solution conditions. X-ray crystallography and transmission electron microscopy (TEM) measurements indicate that all three classes of protein nanotubes are constructed through the same 2D arrangement of Zn(8)R(4) tetramers held together by Zn coordination. The mechanical properties of these nanotubes are correlated with their widths. All Zn(8)R(4) nanotubes are found to be highly flexible despite possessing crystalline order, owing to their small inter-building-block interaction surfaces that are mediated solely by metal coordination.

A broad review of technologically focused work concerning biomolecules at interfaces is presented. The emphasis is on developments in interfacial biomolecular engineering that may have a practical impact in bioanalysis, tissue engineering, emulsion processing or bioseparations. We also review methods for fabrication in an attempt to draw out those approaches that may be useful for product manufacture, and briefly review methods for analysing the resulting interfacial nanostructures. From this review we conclude that the generation of knowledge and-innovation at the nanoscale far exceeds our ability to translate this innovation into practical outcomes addressing a market need, and that significant technological challenges exist. A particular challenge in this translation is to understand how the structural properties of biomolecules control the assembled architecture, which in turn defines product performance, and how this relationship is affected by the chosen manufacturing route. This structure-architecture-process-performance (SAPP) interaction problem is the familiar laboratory scale-up challenge in disguise. A further challenge will be to interpret biomolecular self- and directed-assembly reactions using tools of chemical reaction engineering, enabling rigorous manufacturing optimization of self-assembly laboratory techniques. We conclude that many of the technological problems facing this field are addressable using tools of modem chemical and biomolecular engineering, in conjunction with knowledge and skills from the underpinning sciences. (c) 2005 Elsevier Ltd. All rights reserved.

The stringent processes of natural selection and evolution have enabled extraordinary structure–function properties of biomolecules. Specifically, the archetypal designs of biomolecules, such as amino acids, nucleobases, carbohydrates and lipids amongst others, encode unparalleled information, selectivity and specificity. The integration of biomolecules either with functional molecules or with an embodied functionality ensures an eclectic approach for novel and advanced nanotechnological applications ranging from electronics to biomedicine, besides bright prospects in systems chemistry and synthetic biology. Given this intriguing scenario, our feature article intends to shed light on the emerging field of functional biomolecular engineering.

Protein drugs have revolutionized the pharmaceutical industry, offering new treatments for serious diseases. Since the first recombinant protein drug, Eli Lilly’s Humulin®, was approved 30 years ago (1), protein drugs have grown from esoteric specialty products to a major drug class. Of the 20 top selling drugs in the USA in the third quarter of 2012, nine are proteins. Despite these successes, the inherent instability of protein molecules remains an impediment to their development and to their safety and efficacy. One of the most serious types of instability is aggregation, the self-association of native protein through covalent and/or non-covalent interactions. Protein aggregates have been associated with increased or decreased drug potency and with an increased potential for immunogenic side effects, which can be life-threatening. In this themed issue of the AAPS Journal, we have assembled research and review articles that address the aggregation of therapeutic proteins. The issue was inspired by presentations at the 10th Annual Garnet E. Peck Symposium in Industrial Pharmacy, held at Purdue University in West Lafayette, Indiana, on October 11, 2012. At a practical level, interest in the aggregation of therapeutic proteins is driven by the need for stable formulations and robust manufacturing conditions. At the symposium, Dr. David Volkin (Dept. of Pharmaceutical Chemistry, University of Kansas) presented a series of case studies based on his recent work that address these issues. The cases showed the effects of excipients on the aggregation of an albumin fusion protein (2) and IgG2 monoclonal antibodies (mAbs) (3), the role of infusion bags in the solubility and aggregation of IgG4 mAbs (4), methods to ensure comparability during mAb process development (5), and an empirical phase diagram approach to identifying formulations that inhibit aggregation (6). In their review article in this special issue, Dr. Volkin and his coauthors summarize these case studies, present an overview of protein aggregation mechanisms, and describe high throughput approaches to monitoring protein stability (7). In developing drug products, the industry makes use of accelerated stability studies to estimate shelf life at the storage temperature based on degradation rates measured at higher temperatures. This extrapolation usually assumes that the temperature dependence of reaction rates follows Arrhenius behavior. However, protein aggregation often exhibits non-Arrhenius temperature dependence, even over relatively narrow temperature ranges. At the symposium, Dr. Chris Roberts (Dept. of Chemical and Biomolecular Engineering, University of Delaware) presented his group’s recent work on the mechanisms of protein aggregation and origins of non-Arrhenius behavior (8,9). In this themed issue, he and Dr. Wei Wang (Pfizer BioTherapeutics) summarize these mechanistic insights and discuss the implications for accelerated stability testing (10). Ensuring that protein drugs are aggregate-free requires robust, reproducible analytical methods. The ideal method would resolve aggregate and monomeric species, quantify aggregate size and concentration, and provide a low limit of detection, all while achieving high throughput at moderate cost. Current methods fall short of this ideal. For example, size exclusion chromatography (SEC) assays for soluble aggregates are expensive and low-throughput. Gel electrophoresis (e.g., SDS-PAGE) gives somewhat higher throughput but generally is not quantitative. To address these limitations, Dr. Mary Wirth (Dept. of Chemistry, Purdue University) and her colleagues are developing novel chromatographic materials based on silica colloidal crystals. These ordered arrays of silica particles provide plate heights in the low nanometer range, allowing for high resolution and rapid analysis times. For example, using a capillary packed with silica colloidal crystals and pressure-driven flow, the group has separated a monoclonal antibody and its aggregates in less than a minute with baseline resolution (11). Dr. Wirth and her coauthors summarize the group’s recent results in this themed issue (12). Together, these articles provide insight into our current understanding of protein aggregation. Gaps in fundamental understanding and applied technology remain, however, impeding our ability to monitor and inhibit aggregation in biologics. These gaps include limited understanding of the relative importance of partial unfolding, colloidal interactions, and chemical reaction in the protein aggregation process; a lack of understanding of the chemical and physical determinants of the immune response to protein drugs and their aggregated forms; poor agreement among available analytical methods for determining aggregate size and concentration, particularly in the subvisible range; and an incomplete understanding of the mechanistic effects of process stresses and formulation variables. We hope that the practical and theoretical perspectives assembled here will stimulate additional discussion and research in this important area.

During the next decade or so, there will be significant and impressive advances in biomolecular engineering, especially in our understanding of the biological roles of various biomolecules inside the cell. The advances in high throughput screening technology for discovery of target molecules and the accumulation of functional genomics and proteomics data at accelerating rates will enable us to design and discover novel biomolecules and proteins on a rational basis in diverse areas of pharmaceutical, agricultural, industrial, and environmental applications. As an applied molecular evolution technology, DNA shuffling will play a key role in biomolecular engineering. In contrast to the point mutation techniques, DNA shuffling exchanges large functional domains of sequences to search for the best candidate molecule, thus mimicking and accelerating the process of sexual recombination in the evolution of life. The phage-display system of combinatorial peptide libraries will be extensively exploited to design and create many novel proteins, as a result of the relative ease of screening and identifying desirable proteins. Even though this system has so far been employed mainly in screening the combinatorial antibody libraries, its application will be extended further into the science of protein-receptor or protein-ligand interactions. The bioinformatics for genome and proteome analyses will contribute substantially toward ever more accelerated advances in the pharmaceutical industry. Biomolecular engineering will no doubt become one of the most important scientific disciplines, because it will enable systematic and comprehensive analyses of gene expression patterns in both normal and diseased cells, as well as the discovery of many new high-value molecules. When the functional genomics database, EST and SAGE techniques, microarray technique, and proteome analysis by 2-dimensional gel electrophoresis or capillary electrophoresis in combination with mass spectrometer are all put to good use, biomolecular engineering research will yield new drug discoveries, improved therapies, and significantly improved or new bioprocess technology. With the advances in biomolecular engineering, the rate of finding new high-value peptides or proteins, including antibodies, vaccines, enzymes, and therapeutic peptides, will continue to accelerate. The targets for the rational design of biomolecules will be broad, diverse, and complex, but many application goals can be achieved through the expansion of knowledge based on biomolecules and their roles and functions in cells and tissues. Some engineered biomolecules, including humanized Mab's, have already entered the clinical trials for therapeutic uses. Early results of the trials and their efficacy are positive and encouraging. Among them, Herceptin, a humanized Mab for breast cancer treatment, became the first drug designed by a biomolecular engineering approach and was approved by the FDA. Soon, new therapeutic drugs and high-value biomolecules will be designed and produced by biomolecular engineering for the treatment or prevention of not-so-easily cured diseases such as cancers, genetic diseases, age-related diseases, and other metabolic diseases. Many more industrial enzymes, which will be engineered to confer desirable properties for the process improvement and manufacturing of high-value biomolecular products at a lower production cost, are also anticipated. New metabolites, including novel antibiotics that are active against resistant strains, will also be produced soon by recombinant organisms having de novo engineered biosynthetic pathway enzyme systems. The biomolecular engineering era is here, and many of benefits will be derived from this field of scientific research for years to come if we are willing to put it to good use.

Genetically modified (GM) technique, one of the modern biomolecular engineering technologies, has been deemed as profitable strategy to fight against global starvation. Yet rapid and reliable analytical method is deficient to evaluate the quality and potential risk of such resulting GM products. We herein present a biomolecular analytical system constructed with distinct biochemical activities to expedite the computational detection of genetically modified organisms (GMOs). The computational mechanism provides an alternative to the complex procedures commonly involved in the screening of GMOs. Given that the bioanalytical system is capable of processing promoter, coding and species genes, affirmative interpretations succeed to identify specified GM event in terms of both electrochemical and optical fashions. The biomolecular computational assay exhibits detection capability of genetically modified DNA below sub-nanomolar level and is found interference-free by abundant coexistence of non-GM DNA. This bioanalytical system, furthermore, sophisticates in array fashion operating multiplex screening against variable GM events. Such a biomolecular computational assay and biosensor holds great promise for rapid, cost-effective, and high-fidelity screening of GMO.

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods(1-20) wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize. In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges(21-23) and (b) grippers that self-fold due to residual stress powered hinges(24,25). The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales(22, 26) and with a range of materials including metals(21), semiconductors(9) and polymers(27). With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 μm to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films(5,7) to possibly create even smaller nanoscale grasping devices.

Human beta defensins (hBDs) are natural antimicrobial peptides (AMPs) with broad spectrum antimicrobial activity. However, hBDs, like many AMPs, are easily inactivated by salt, which limits their extracellular applications as antimicrobial coating agents. In this study, a salt-resistant hBD28 peptide was designed by increasing C-terminus cationicity of the wild type peptide via rational amino acid substitution. The mutant hBD28 exhibited salt-tolerance behaviour and improved antimicrobial potency compared to wild type hBD28. Zeta potential analysis confirmed that increased cationicity was crucial to overcome salt-induced charge-shielding effects, which enhanced peptide–membrane interaction compared to the wild type peptide. The mutant hBD28 did not exhibit obvious differences with respect to hydrophobicity, oligomerization ability, and secondary structure compared to the wild type peptide. A simple design strategy to overcome salt-inactivation in hBD28 is demonstrated through this study, which will guide the design of other salt-resistant AMPs to accelerate their development as anti-infective agents in ionic environments.

Nanomedicine is an emerging field that integrates nanotechnology, biomolecular engineering, life sciences and medicine; it is expected to produce major breakthroughs in medical diagnostics and therapeutics. Due to the size-compatibility of nano-scale structures and devices with proteins and nucleic acids, the design, synthesis and application of nanoprobes, nanocarriers and nanomachines provide unprecedented opportunities for achieving a better control of biological processes, and drastic improvements in disease detection, therapy, and prevention. Recent advances in nanomedicine include the development of functional nanoparticle based molecular imaging probes, nano-structured materials as drug/gene carriers for in vivo delivery, and engineered molecular machines for treating single-gene disorders. This review focuses on the development of molecular imaging probes and engineered nucleases for nanomedicine, including quantum dot bioconjugates, quantum dot-fluorescent protein FRET probes, molecular beacons, magnetic and gold nanoparticle based imaging contrast agents, and the design and validation of zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs) for gene targeting. The challenges in translating nanomedicine approaches to clinical applications are discussed.

We review simulation and experiments using protein microarrays. Arrays of thousands of proteins with varied functionalities offer parallel, high throughput screening of molecular interactions. Immobilization of probe molecules to a surface or bead allows for location to be equated with identification. The inhomogeneity present because of the material surface can alter the thermodynamic and kinetic recognition properties of the proteins. Understanding the behavior of proteins at interfaces has implications in the design of protein microarrays as well as in the development of other interfacial biomolecular engineering technologies. In this review, we describe current problems when immobilizing peptides/proteins on material surfaces. The interface between experimental and simulation studies is discussed regarding orientation, and conformational stability on materials with varied surface chemistry in terms of protein-surface interactions.

Cell adhesion to matrix, other cells, or pathogens plays a pivotal role in many processes in biomolecular engineering. Early macroscopic methods of quantifying adhesion led to the development of quantitative models of cell adhesion and migration. The more recent use of sensitive probes to quantify the forces that alter or manipulate adhesion proteins has revealed much greater functional diversity than was apparent from population average measurements of cell adhesion. This review highlights theoretical and experimental methods that identified force-dependent molecular properties that are central to the biological activity of adhesion proteins. Experimental and theoretical methods emphasized in this review include the surface force apparatus, atomic force microscopy, and vesicle-based probes. Specific examples given illustrate how these tools have revealed unique properties of adhesion proteins and their structural origins.

Don't forget poly(disulfide)s. There is a rich literature pointing out the advantages of the dynamic nature of single disulfide bridges to explore self-sorting, biomolecular engineering, biomembrane analysis, and so on. Disulfide bonds between polymer chains are essential for protein folding, materials properties and the stabilization of various supramolecular architectures. However, poly(disulfide)s with disulfide bonds in the main chain are rarely used today to create interesting structures or functions. To attract attention and outline scope and limitations of poly(disulfide)s to build modern supramolecular systems, the rather eclectic recent literature on the topic is summarized. The review is moving from fascinating basic studies including photoinduced metathesis, polycatenanes and polyrotaxanes to applications in biosupramolecular systems such as micelles, membranes, tubes, gels, carriers, pores, sensors, catalysts and photosystems.

Constructing the next generation of optical, electronic, and magnetic materials requires creating organized, defect-free structures on a nanoscale. In his perspective, Ober discusses new work ( Belcher et al.) that merges biomolecular engineering with materials science to create a nanocomposite of bacteriophage and ZnS nanoparticles. The nanoparticles occupy narrow layers periodically spaced by long viral rods.