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Erratum Erratum to ‘‘Pharmacophore modeling strategies for the development of novel nonsteroidal inhibitors of human aromatase (CYP19)” [Bioorg. Med. Chem. Lett. 20 (2010) 3050] Yagmur Muftuoglu , Gabriela Mustata b,* Departments of Biophysics and Chemical Biomolecular Engineering, Johns Hopkins University, Baltmore, MD 21218, United States Department of Computational Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, United States

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.

A review of current processing practices in preparation of recombinant single chain antibody fragments is presented. Single chain antibody fragments which are superior to their Fab and IgG counterparts due to their higher affinity for target antigens while imposing minimal antigenicity in recipient hosts, have sparked breakthroughs in immunology and the medical field at large. The rapidly increasing market demand for pure single chain antibodies for research and therapeutic applications, necessitates viable manufacture routes that can produce large amounts of these antibodies efficiently and as cheaply as possible. Medium- to high-producing expression systems reported for recombinant single chain antibody production are reviewed, and their reported or potential success for efficient commercial-viable preparation of pure antibodies discussed. The effects of expression host system choice on product molecular constraints, ease of processing, and flowsheet design and scale-up are compared. It is concluded that there is no unique host system that can consistently yield high expression levels for a wide range of single chain antibodies; instead, product sequence and end application often dictate the optimum choice of expression host. Irrespective of host systems, adequate a priori design and engineering of the molecular construct supported by good biophysical understanding of the single chain fragment molecules, is a crucial pre-requisite for improved product stability and downstream recovery, which will favourably impact final product yield and functionality.

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.

Peptides with the cystine-knot architecture, often termed knottins, are promising scaffolds for biomolecular engineering. These unique molecules combine diverse bioactivities with excellent structural, thermal, and proteolytical stability. Being different in the composition and structure of their amino acid backbone, knottins share the same core element, namely cystine knot, which is built by six cysteine residues forming three disulfides upon oxidative folding. This motif ensures a notably rigid framework that highly tolerates both rational and combinatorial changes in the primary structure. Being accessible through recombinant production and total chemical synthesis, cystine-knot miniproteins can be endowed with novel bioactivities by variation of surface-exposed loops and incorporation of non-natural elements within their non-conserved regions towards the generation of tailor-made peptidic compounds. In this chapter the topology of cystine-knot peptides, their synthesis and applications for diagnostics and therapy is discussed.

Living organisms have been exploited as production hosts for a large variety of compounds. To improve the efficiency of bioproduction, metabolic pathways in an organism are usually manipulated by various genetic modifications. However, bottlenecks during the conversion of substrate to a desired product may result from cellular regulations at different levels. Dynamic regulation of metabolic pathways according to the need of cultivation process is therefore essential for developing effective bioprocesses, but represents a major challenge in metabolic engineering and synthetic biology. To this end, switchable biomolecules which can sense the intracellular concentrations of metabolites with different response types and dynamic ranges are of great interest. This chapter summarizes recent progress in the development of biomolecular switches and their applications for improvement of bioproduction via dynamic control of metabolic fluxes. Further studies of bioswitches and their applications in industrial strain development are also discussed.

The activity of protein kinases is heavily dependent on the phosphorylation state of the protein. Kinase phosphorylation states have been prepared through biological or enzymatic means for biochemical evaluation, but the use of protein chemical modification as an investigative tool has not been addressed. By chemically reacting a genetically encoded cysteine, phosphocysteine was installed via dehydroalanine as a reactive intermediate. The installed phosphocysteine was intended as a surrogate to the naturally occurring phosphothreonine or phosphoserine of a phosphorylated protein kinase. Two model protein kinases were investigated on: MEK1 and p38α. The development of suitable protein variants and suitable reaction conditions on these two proteins is discussed in turn and in detail, resulting in p38α-pCys180 and MEK1-pCys222. Designed to be mimics of the naturally occurring p38α-pThr180 and MEK1-pSer222, these two chemically modified proteins were studied for their biological function. The core biological studies entailed the determination of enzymatic activity of both modified proteins, and included the necessary controls against their active counterparts. In addition, the studies on p38α-pCys180 also included a more detailed quantification of enzymatic activity, and the behaviour of this modified protein against known inhibitors of p38α was also investigated. Both modified proteins were shown to be enzymatically active and behave similarly to corresponding active species. The adaptation of mass spectrometry methods to handle the majority of project's analytical requirements, from monitoring chemical transformations to following enzyme kinetics was instrumental in making these studies feasible. The details of these technical developments are interwoven into the scientific discussion. Also included in this thesis is an introduction to the mechanism and function of protein kinases, and on the protein chemistry methods employed. The work is concluded with a projection of implications that this protein chemical modification technique has on kinase biomedical research.

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.

Understanding the microscopic structure and macroscopic properties of condensed matter from a molecular perspective is important for both traditional and modern chemical engineering. A cornerstone of such understanding is provided by statistical mechanics, which bridges the gap between molecular events and the structural and physiochemical properties of macro- and mesoscopic systems. With ever-increasing computer power, molecular simulations and ab initio quantum mechanics are promising to provide a nearly exact route to accomplishing the full potential of statistical mechanics. However, in light of their versatility for solving problems involving multiple length and timescales that are yet unreachable by direct simulations, phenomenological and semiempirical methods remain relevant for chemical engineering applications in the foreseeable future. Classical density functional theory offers a compromise: on the one hand, it is able to retain the theoretical rigor of statistical mechanics and, on the other hand, similar to a phenomenological method, it demands only modest computational cost for modeling the properties of uniform and inhomogeneous systems. Recent advances are summarized of classical density functional theory with emphasis on applications to quantitative modeling of the phase and interfacial behavior of condensed fluids and soft materials, including colloids, polymer solutions, nanocomposites, liquid crystals, and biological systems. Attention is also given to some potential applications of density functional theory to material fabrications and biomolecular engineering. © 2005 American Institute of Chemical Engineers AIChE J, 2006

With recent advances in biomolecular engineering, the bioremediation of persistent organic pollutants (POPs) using genetically modified microorganisms has become a rapidly growing area of research for environmental protection. Two main biomolecular approaches, rational design and directed evolution, have been developed to engineer enhanced microorganisms and enzymes for the biodegradation of POPs. This review describes the most recent developments and applications of these biomolecular tools for enhancing the capability of microorganisms to bioremediate three major classes of POPs – polycyclic aromatic hydrocabons (PAHs), polychlorinated biphenyls (PCBs) and pesticides. Most of the examples focused on the redesign of various features of the enzymes involved in the bioremediation of POPs, including the enzyme expression level, enzymatic activity and substrate specificity. Overall, the rapidly expanding potential of biomolecular engineering techniques has created the exciting potential of remediating some of the most recalcitrant and hazardous compounds in the environment.

Biomolecular engineering is hot, the tech sector is turning around, and the United States will continue to dominate high-tech R&D in the coming decade. So say the IEEE Fellows in the second annual 2004 IEEE Technology Leaders Survey.

A promising alternative to replace the current egg- or cell culture-based technology for vaccine production from live viruses is virus-like particle (VLP) technology based on a microbial platform. VLPs are macromolecular assemblies of viral capsid proteins, which have been shown to tolerate insertion of antigen modules via genetic recombinant technology, yielding modular VLPs. Many studies on modular VLPs presume that when a peptide antigen element is taken out from the intact proteins and then modularised on VLPs, it is unable to fold into its native structure. However, until now, presentation of a peptide antigen element on a VLP and the impact of the display strategy to present the antigen element on the quality of the resulting antibodies (i.e. the ability of the antibodies to recognise the intact protein) are not fully understood. This thesis aims to understand the underlying fundamentals regarding modularisation of peptide antigen elements on VLPs for induction of high-quality antibodies. A hypervariable receptor-binding domain, Helix 190 (H190), from the hemagglutinin protein of influenza A virus was used as a model for modularisation on VLPs from murine polyomavirus (MuPyV) VP1 protein. Four major findings are presented. Firstly, two display strategies, i.e. arraying of H190 in tandem repeats and the use of helix promoter elements, were shown to display H190 in its immunogenic form equally. However, modularisation using tandem repeat display induced antibodies of a higher quality than modularisation using helix promoter elements. Secondly, the quality of antibodies induced by the tandem repeat display bearing two copies of H190 was optimum, thus no significant improvement was observed following the use of adjuvant or increasing the copy number of H190. Additionally, the increase in the copy number of H190 was shown to reduce the assembly capability and solubility of modular VP1 in an environment that was optimised for wild-type VP1. Thirdly, this thesis shows the novel finding in the use of flanking ionic elements to stabilise VLP precursors, termed as capsomeres, bearing two copies of H190 containing a hydrophobic stretch, which caused aggregation. Fourthly, the first steps towards obtaining the atomic crystal structure of presented H190 on a modular protein were performed, i.e. a mild and satisfactory laboratory process was developed to achieve high-purity modular VP1 capsomeres, unattainable using previously established expression and purification process of wild-type MuPyV VP1. This thesis shows a step forward towards understanding the presentation of a peptide antigen element on a VLP that enables induction of highquality antibodies, and towards VLP engineering to manipulate the aggregation and solubility of modular VP1. VLP technology based on a microbial platform presented here is a potentially safe and effective alternative vaccine candidate that targets a hypervariable peptide antigen element. The speed of the microbial platform allows a rapid response to the hypervariability of the peptide antigen element, which otherwise may be unachievable using the egg- and cell culture-based technologies.

Microbial transglutaminase (MTG), a robust enzyme developed initially for the manipulation of edible proteins in the food industry, has now been widely recognized as a practical protein-modifying reagent in the range of biotechnological applications. In this chapter, we introduce the potential use of MTG through our basic studies on the design of novel glutamine (Gln) donor substrates for lysine (Lys)-specific protein modification. Based on the core structure of a conventional transglutaminase substrate, benzyloxycarbonyl-L-glutaminylglycine (Z-QG), new Gln-donor substrates have been developed for the conjugation of recombinant proteins with different functionalities. The first target site for the substrate engineering was the C-terminal carboxylic group of Z-QG, which is feasibly labeled with functional moieties. For the preparation of protein-nucleic acid conjugates with novel molecular architecture, a new nucleotidyl substrate, Z-QG-(d)UTP, was created. We have also explored substitution of the N-terminal protecting group (Z) with fluorophores and biotin, and found that MTG accepts diverse functional groups at the N-terminus by inserting a short linker, leading to an increase in the utility of MTG in site-specific modification of functional proteins. Our results demonstrated how the design of (small) Gln-donor substrates of MTG can expand the scope of enzymatic manipulation in biomolecular engineering.

1 School of Environmental and Chemical Engineering, Yanshan University, Qinhuangdao 066004, China 2 Institute of Materials Science of Mulhouse (IS2M), 15 Rue Jean Starcky, P.O. Box 2488, 68057 Mulhouse Cedex, France 3 Department of Chemical and Biomolecular Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong 4Department of Environmental Engineering, Yanshan University, Qinhuangdao 066004, China

Antibodies are important molecules in many research fields, where they play a key role in various assays. Antibody labeling is therefore of great importance. Currently, most labeling techniques take advantage of certain amino acid side chains that commonly appear throughout proteins. This makes it hard to control the position and exact degree of labeling of each antibody. Hence, labeling of the antibody may affect the antibody-binding site. This paper presents a novel protein domain based on the IgG-binding domain C2 of streptococcal protein G, containing the unnatural amino acid BPA, that can cross-link other molecules. This novel domain can, with improved efficiency compared to previously reported similar domains, site-specifically cross-link to IgG at the Fc region. An efficient method for simultaneous in vivo incorporation of BPA and specific biotinylation in a flask cultivation of Escherichia coli is described. In comparison to a traditionally labeled antibody sample, the C2-labeled counterpart proved to have a higher proportion of functional antibodies when immobilized on a solid surface and the same limit of detection in an ELISA. This method of labeling is, due to its efficiency and simplicity, of high interest for all antibody-based assays where it is important that labeling does not interfere with the antibody-binding site.

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.

Natural selection has created optimal catalysts that exhibit their convincing performance even with a number of sometimes counteracting constraints. Optimal performance of enzyme catalysis does not refer necessarily to maximum reaction rate. Rather, it may involve a compromise between specificity, rate, stability, and other chemical constraints ; in some cases, it may involve aintelligento control of rate and specificity. Because enzymes are capable of catalyzing reactions under mild conditions and with high substrate specificity that often is accompanied by high regioand enantioselectivity, it is not surprising that a continually increasing number of industrial and academic reports concern the use of enzyme catalysts in chemical synthesis as well as in biochemical and biomedical applications. However, the demands of modern synthesis and their commercial application were obviously not targeted during the natural evolution of enzymes. Considering a specific, nonnatural application, any property (or combination of properties) of an enzyme may therefore need to be improved. Of course, scientists desired to mimick nature's powerful concepts for tailoring specific enzymatic properties: Following pioneering experiments for evolving molecules in the test tube, evolutionary engineering of biomolecules was successfully realized with first selections of functional nucleic acids (ribozymes) by using the SELEX (systematic evolution of ligands by exponential enrichment with integrated optimization by non-linear analysis) procedure, 8] and with the development of high-affinity ligands (aptamers) by using similar techniques. Meanwhile, evolutionary engineering, also termed adirected evolutiono, has emerged as a key technology for biomolecular engineering and generated impressive results in the functional adaptation of enzymes to artificial environments. Certainly, evolution in the laboratory does not come to a halt at the optimization of single genes and proteins. Recent results excitingly demonstrate the success of amolecular breedingo of metabolic pathways, and even of complete genomes, and the end is not in sight yet. Directed evolution in the laboratory is highly attractive because its principles are simple and do not require detailed knowledge of structure, function, or mechanism. Essentially like natural evolution, directed evolution comprises the iterative implementation of (1) the generation of a alibraryo of mutated genes, (2) its functional expression, and (3) a sensitive assay to identify individuals showing the desired properties, either by selection or by screening (Figure 1). After each round, the genes of improved variants are deciphered and subsequently serve as parents for another round of optimization. This review covers the most important aspects of directed evolution and summarizes key solutions to problems of optimizing and understanding enzyme function.

The advances in high throughput screening technology for discovery of target molecules and the accumulation of functional genomics and proteomics data at an ever-accelerating rate 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. The biomolecular engineering will no doubt become one of the most important scientific disciplines in that it will enable us to comprehensively analyze gene expression patterns in both normal and diseased cells and to discover many new biologically active molecules rationally and systematically. 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 more novel proteins, due to the relative ease of screening and identifying desirable proteins. Its application will be extended further into the science of protein–receptor or protein–ligand interactions. The bioinformatics including EST-based or SAGE-tag-based functional genomics and proteomics will continue to advance rapidly. Its biological knowledge base will expand the scope of biomolecular engineering, and the impact of well-coordinated biomolecular engineering research will be very significant on our understanding of gene expression, upregulation and downregulation, and posttranslational protein processing in healthy and diseased cells. The bioinformatics for genome and proteome analysis will contribute substantially toward ever more accelerated advances in pharmaceutical industry. When the functional genomics database, EST and SAGE techniques, microarray technique, and proteome analysis by 2-dimensional gel electrophoresis or capillary electrophoresis are all put to good use, the biomolecular engineering research will yield new drug discoveries, improved therapies, and new or significantly improved bioprocesses. 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 be accelerated. The targets for rational design of biomolecules will be very broad, diverse, and complex, but many application goals can be achieved through the expansion of knowledge base on biomolecules of interest and their roles and functions in cells and tissues. In the near future, more therapeutic drugs and high-value biomolecules will be designed and produced for the treatment or prevention of not-so-easily-cured diseases such as cancers, genetic diseases, age-related diseases, and other metabolic diseases. Also anticipated are many more industrial enzymes that will be engineered to confer desirable properties for the process improvement and manufacturing of many high-value biomolecular products. Many more new metabolites including novel antibiotics that are active against resistant strains will be also produced by recombinant organisms having de novo engineered biosynthetic pathway enzyme systems. The biomolecular engineering era is here and a great deal of benefits can be derived from this field of scientific research for many years to come if we are willing to put it to good use.

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.

Biomolecular engineering is a technology to create novel structures of high-value biomolecules for use in medicine and industry, through the directed alteration of proteins and/or biologically active molecules in living cells to produce a novel biometabolites as well as engineered protein itself. For the development of new drugs by biomolecular engineering, desired biomolecules have to be rationally designed based on their structure-stability/structure-activity relationship, and then screened through well-established mutation and selection program. Over the past decade, there has been significant progress in mutation and selection methodology; DNA shuffling technology mimicking natural evolution for artificial DNA recombination and phage-displayed combinatorial peptide library for rapid selection of proteins expressed from mutated genes. Bioinformatic tools including functional genomics and proteomics have been also developed for the ready access to the information related to the protein-function and genome-protein, leading to the design and identification of new drug targets. Throughout the use of an enormous amount of bioinformatic databases, many protein/peptide drugs and biometabolite molecules have been designed. The candidates of new drugs are monoclonal antibodies, vaccines, enzymes, antibiotics, therapeutic peptides, and so on. Two humanized monoclonal antibodies approved by FDA became the first line of drugs designed by biomolecular engineering approach. They are Herceptin and Synagis, for the treatment of breast cancer and pediatric respiratory syncytial viral infection, respectively. Many more newly engineered biomolecules are under developing for medicinal application. Some clinical trials for therapeutic applications are now in progress, and very positive results are already anticipated.

Huimin Zhao is a professor of Chemical and Biomolecular Engineering at the University of Illinois at Urbana-Champaign and the holder of Centennial Chair. His research interests encompass many different aspects of synthetic biology and chemical biology. They include directed evolution and rational design of proteins and pathways, novel approaches for protein, pathway, and genome engineering, the molecular genetics and enzymology of natural product biosynthesis, the fundamental aspects of biocatalysis, and the use of protein engineering and metabolic engineering methods in designing platform organisms including E. coli and S. cerevisiae for the synthesis of important chemicals, antibiotics, and biofuels from renewable feedstocks.

1 Division of Green Chemistry and Engineering Research, Korea Research Institute of Chemical Technology (KRICT), Daejeon 305-600, Republic of Korea 2 Chemical and Biomolecular Engineering, Clemson University, Clemson, SC 29634, USA 3Department of Materials Science and Technology, College of Engineering, University of Fukui, Fukui 910-8507, Japan 4Department of Biological & Chemical Engineering, Inha University, Incheon 402-751, Republic of Korea

Chemical engineering as a new engineering and science field developed during and after the Second World War, and has had a major impact on global technology and economy. Its development has been greatly accelerated with use of computers, mathematical software, and later internet. Success of the new field was followed with intensive publication activity of university level textbooks, engineering manuals, various data books and numerous scientific and professional journals. This trend has been followed in Croatia by journal Kemija u industriji (Chemistry in industry) which was devoted to transfer needed specific knowledge from international journals to engineers in Croatian language and also report on development and results of chemical engineers in industry and academia in Croatia. Some 40 years ago, at that time young, several chemical engineers from University of Zagreb returned from their doctoral studies from USA and together with their colleagues from Austria, Italy and Slovenia initiated publication in Zagreb a new international journal Chemical and Biochemical Engineering Quarterly (CABEQ) with aim to follow the major trend which started in american departments and present in publications and also begin to develop in Europe. At that time CABEQ was one of few european journals with content in the two seemingly, at that time, unrelated fields. From the very first issue CABEQ was covered in Chemical Abstract, and soon in SCOPUS and Web of Science. Being open access journal it is now present in numerous open access web sites in Europe, Asia and Africa. For the first 10 years the two fields became very successful in application of classical chemical engineering methodology and industrial microbiology resulting in improved production of bulk chemical and biopharmacetical products. Most of manuscripts published in that period excelled in application of mathematical methodology applied to industrial microbiology with aim to increase productivity, process scale-up, advanced process control and maximisation of product quality. However, at that time, most of the inovative ideas were from modern engineering fields applied to new bioprocesses. The trend has greatly changed some 20 years ago with advancement of genetic engineering. Basical new ideas were now brought into the field from biology, rather than from technical fields. Also the focus has been redirected from bulk chemicals to broad aspects of biotechnology aimed to environment protection and biofuels (biorefineries). For about the last 5 years CABEQ journal also reflects the latest trends in biomolecular engineering, focused on improved pharmaceuticals, microtechnology, and medical applications.

Interest in noncovalent interactions involving halogens, particularly halogen bonds (X-bonds), has grown dramatically in the past decade, propelled by the use of X-bonding in molecular engineering and drug design. However, it is clear that a complete analysis of the structure-energy relationship must be established in biological systems to fully exploit X-bonds for biomolecular engineering. We present here the first comprehensive experimental study to correlate geometries with their stabilizing potentials for fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) X-bonds in a biological context. For these studies, we determine the single-crystal structures of DNA Holliday junctions containing halogenated uracil bases that compete X-bonds against classic hydrogen bonds (H-bonds), estimate the enthalpic energies of the competing interactions in the crystal system through crystallographic titrations, and compare the enthalpic and entropic energies of bromine and iodine X-bonds in solution by differential scanning calorimetry. The culmination of these studies demonstrates that enthalpic stabilization of X-bonds increases with increasing polarizability from F to Cl to Br to I, which is consistent with the σ-hole theory of X-bonding. Furthermore, an increase in the X-bonding potential is seen to direct the interaction toward a more ideal geometry. However, the entropic contributions to the total free energies must also be considered to determine how each halogen potentially contributes to the overall stability of the interaction. We find that bromine has the optimal balance between enthalpic and entropic energy components, resulting in the lowest free energy for X-bonding in this DNA system. The X-bond formed by iodine is more enthalpically stable, but this comes with an entropic cost, which we attribute to crowding effects. Thus, the overall free energy of an X-bonding interaction balances the stabilizing electrostatic effects of the σ-hole against the competing effects on the local structural dynamics of the system.

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.

The increasing amount of pollutants in the environment is an alarming concern to the ecosystem. A number of organic pollutants, such as polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), and pesticides, are resistant to degradation, which represent toxological threat to wildlife as well as human beings. Various physiological and biological measures have been employed globally to degrade these hydrocarbons to improve environment quality. Out of these, bioremediation is the most promising strategy where microorganisms are harnessed to degrade the organic and inorganic pollutants. There are many naturally existing microbes, which are routinely employed in bioremediation process. At instances, these consortia of microorganisms in various environmental conditions provide an insight about the interrelation of metabolic pathways involved in biodegradation process. Various metabolic techniques are employed to produce genetically engineered microorganisms (GEMs) with better bioremediation efficiency. Majorly biomolecular engineering approaches such as rational designing and directed evolution have been developed to genetically modify microorganisms and their enzymes for the degradation of persistent organic pollutants (POPs) like PAHs, PCBs, and pesticides. Recently, several developments in the field of recombinant DNA technologies such as development of “suicidal-GEMs” (S-GEMs) have also been carried out to achieve safe and efficient bioremediation of contaminated sites. In this chapter, we describe various techniques for the development of genetically modified microorganisms along with different examples of recombinant produced. Harmful impact of the engineered microorganisms on environment and economic consideration of viable processes development are critically discussed.

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.

An interactive, multidisciplinary, public workshop, organized by a group of experts in biochemistry, biophysics, chemical and biomolecular engineering, chemistry, microbial metabolism, and protein structure and function, was held on January 6-7, 2011 in Washington, DC. Fundamental insights into the biological energy capture, storage, and transformation processes provided by speakers was featured in this workshop which included topics such as microbes living in extreme environments such as hydrothermal vents or caustic soda lakes (extremophiles) provided a fascinating basis for discussing the exploration and development of new energy systems. Breakout sessions and extended discussions among the multidisciplinary groups of participants in the workshop fostered information sharing and possible collaborations on future bioinspired research. Printed and web-based materials that summarize the committee's assessment of what transpired at the workshop were prepared to advance further understanding of fundamental chemical properties of biological systems within and between the disciplines. In addition, webbased materials (including two animated videos) were developed to make the workshop content more accessible to a broad audience of students and researchers working across disciplinary boundaries. Key workshop discussion topics included: Exploring and identifying novel organisms; Identifying patterns and conserved biological structures in nature; Exploring and identifying fundamental properties and mechanisms of knownmore » biological systems; Supporting current, and creating new, opportunities for interdisciplinary education, training, and outreach; and Applying knowledge from biology to create new devices and sustainable technology.« less

The biological production of fuels from renewable sources has been regarded as a feasible solution to the energy and environmental problems in the foreseeable future. Recently, the biofuel product spectrum has expanded from ethanol and fatty acid methyl esters (biodiesel) to other molecules, such as higher alcohols and alkanes, with more desirable fuel properties. In general, biosynthesis of these fuel molecules can be divided into two phases: carbon chain elongation and functional modification. In addition to natural fatty acid and isoprenoid chain elongation pathways, keto acid-based chain elongation followed by decarboxylation and reduction has been explored for higher alcohol production. Other issues such as metabolic balance, strain robustness, and industrial production process efficiency have also been addressed. These successes may provide both scientific insights into and practical applications toward the ultimate goal of sustainable fuel production.

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.

Food materials science is an area of rapid growth in the larger field of food science. It is an applied area overlapping food science and food engineering that is concerned with structure, properties and processing of te materials used in food as well as their production and breakdown. The goals of researchers working in this area include: measuring the size and distribution of elements in a complex mixture, understanding how these elements interact and using this information to develop processes that will maximize the formation of useful structures. Examples are provided of newer methods for generating these structures such as the use of biomolecular engineering to design and produce novel proteins using amino acid sequences not found in nature. The concept of composite materials has been applied to foods and would appear to offer some advantages in dealing with these complex structures. An example is given dealing with the production of a food material composite, mixed and filled dairy gels, in which casein micelles are absorbed at the surface of protein-coated fat globules, leading to the formation of a copolymer network with overall greater strength. Of equal interest to the development of new food materials is how they behave under mechanical stress, and the idea of fracture behaviour, in particular notch sensitivity, is illustrated.

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.

Department of Chemical & Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576, Singapore and Division of Bioengineering, National University of Singapore, 7 Engineering Drive 1, Singapore 117576, Singapore and Nanoscience & Nanotechnology Initiative, National University of Singapore, 2 Engineering Drive 3, Singapore 117587, Singapore Targeted nanomedicine for detection and treatment of circulating tumor cells

Genetic and biomolecular engineering are two of the leading disciplines in biotechnology that have lead to great advancements in protein engineering. Applications of analytical bioluminescence, such as genetic reporter assays, optical in vivo imaging, and cell viability assays can often be improved by enhancing wild-type bioluminescent systems. The ability to rationally or randomly modify proteins has expanded their employment in various bioanalytical applications. Specifically, a wide range of bioluminescent proteins and photoproteins have been engineered that can be utilized in many detection and diagnostic applications. Herein, we focus on the improvements of two of the most commonly studied photoproteins, aequorin and obelin, and their uses in a variety of bioanalytical applications. Techniques such as random mutagenesis, site-directed mutagenesis, bioluminescence resonance energy transfer, and the incorporation of coelenterazine analogues are discussed as ways that have expanded the palette of these designer proteins by altering their emission wavelengths and/or half-lifes. Strategic amino acid substitutions and insertions have been also used to improve luciferase stability in high temperature, extreme pH, and harsh chemical environments, and to customize their kinetic properties and bioluminescence colors. As researchers advance engineering techniques to expand the array of photoproteins, luciferases from fireflies, click beetles, marine organisms and bacteria, their use in bioanalytical applications will continue to grow and it is envisioned that photoproteins and bioluminescent proteins will become as diverse as their fluorescence counterparts.