Biomolecular engineering and drug development

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.     

Advances in directed protein evolution by recursive genetic recombination: applications to therapeutic proteins

Recent developments in directed evolution technologies combined with innovations in robotics and screening methods have revolutionized protein engineering. These methods are being applied broadly to many fields of biotechnology, including chemical engineering, agriculture and human therapeutics. More specifically, DNA shuffling and other methods of genetic recombination and mutation have resulted in the improvement of proteins of therapeutic interest. Optimizing genetic diversity and fitness through iterative directed evolution will accelerate improvements in engineered protein therapeutics.     

Search and discovery strategies for biotechnology: the paradigm shift.

Profound changes are occurring in the strategies that biotechnology-based industries are deploying in the search for exploitable biology and to discover new products and develop new or improved processes. The advances that have been made in the past decade in areas such as combinatorial chemistry, combinatorial biosynthesis, metabolic pathway engineering, gene shuffling, and directed evolution of proteins have caused some companies to consider withdrawing from natural product screening. In this review we examine the paradigm shift from traditional biology to bioinformatics that is revolutionizing exploitable biology. We conclude that the reinvigorated means of detecting novel organisms, novel chemical structures, and novel biocatalytic activities will ensure that natural products will continue to be a primary resource for biotechnology. The paradigm shift has been driven by a convergence of complementary technologies, exemplified by DNA sequencing and amplification, genome sequencing and annotation, proteome analysis, and phenotypic inventorying, resulting in the establishment of huge databases that can be mined in order to generate useful knowledge such as the identity and characterization of organisms and the identity of biotechnology targets. Concurrently there have been major advances in understanding the extent of microbial diversity, how uncultured organisms might be grown, and how expression of the metabolic potential of microorganisms can be maximized. The integration of information from complementary databases presents a significant challenge. Such integration should facilitate answers to complex questions involving sequence, biochemical, physiological, taxonomic, and ecological information of the sort posed in exploitable biology. The paradigm shift which we discuss is not absolute in the sense that it will replace established microbiology; rather, it reinforces our view that innovative microbiology is essential for releasing the potential of microbial diversity for biotechnology penetration throughout industry. Various of these issues are considered with reference to deep-sea microbiology and biotechnology.     

Biogenesis of the Escherichia coli haemolysin toxin

Pore-forming proteins are being engineered to produce new components for biomolecular materials and devices. The primary target of these studies has been staphylococcal α-haemolysin, which is a 293 amino acid, water soluble polypeptide that self assembles in lipid bilayers to form heptameric pores of 1–2 nm internal diameter. By genetic engineering and targeted chemical modification, we have produced α-haemolysins in which pore activity can be triggered or switched on and off by biochemical, chemical or physical stimuli, including the action of enzymes, non-covalent and covalent modification, and irradiation with near UV light. One application is in sensor technology. The remodelled molecules can be used as components of sensitive, selective, real-time sensors for the stimuli that affect their activities, as demon strated with divalent metal ions as analyte. Now, we are using combinatorial mutagenesis and DNA shuffling, as well as structure-based approaches, to generate homomeric and heteromeric pores with high affinity binding sites for additional analytes.     

Engineering biosynthesis of high-value compounds in photosynthetic organisms

The photosynthetic, autotrophic lifestyle of plants and algae position them as ideal platform organisms for sustainable production of biomolecules. However, their use in industrial biotechnology is limited in comparison to heterotrophic organisms, such as bacteria and yeast. This usage gap is in part due to the challenges in generating genetically modified plants and algae and in part due to the difficulty in the development of synthetic biology tools for manipulating gene expression in these systems. Plant and algal metabolism, pre-installed with multiple biosynthetic modules for precursor compounds, bypasses the requirement to install these pathways in conventional production organisms, and creates new opportunities for the industrial production of complex molecules. This review provides a broad overview of the successes, challenges and future prospects for genetic engineering in plants and algae for enhanced or de novo production of biomolecules. The toolbox of technologies and strategies that have been used to engineer metabolism are discussed, and the potential use of engineered plants for industrial manufacturing of large quantities of high-value compounds is explored. This review also discusses the routes that have been taken to modify the profiles of primary metabolites for increasing the nutritional quality of foods as well as the production of specialized metabolites, cosmetics, pharmaceuticals and industrial chemicals. As the universe of high-value biosynthetic pathways continues to expand, and the tools to engineer these pathways continue to develop, it is likely plants and algae will become increasingly valuable for the biomanufacturing of high-value compounds.     

蛋白质定向进化的研究进展及其应用前景

定向进化是改造蛋白质分子的有效新策略.它不需要了解蛋白质的空间结构,主要通过在实验室里模拟自然进化过程,采用错误倾向PCR等方法对编码蛋白质的基因进行随机突变,经DNA改组、交错延伸等技术进行体外重组,设计高通量筛选方法来选出需要的突变体.本综述了定向进化技术的发展及应用.     

Emerging tools for real-time label-free detection of interactions on functional protein microarrays

The availability of extensive genomic information and content has spawned an era of high-throughput screening that is generating large sets of functional genomic data. In particular, the need to understand the biochemical wiring within a cell has introduced novel approaches to map the intricate networks of biological interactions arising from the interactions of proteins. The current technologies for assaying protein interactions--yeast two-hybrid and immunoprecipitation with mass spectrometric detection--have met with considerable success. However, the parallel use of these approaches has identified only a small fraction of physiologically relevant interactions among proteins, neglecting all nonprotein interactions, such as with metabolites, lipids, DNA and small molecules. This highlights the need for further development of proteome scale technologies that enable the study of protein function. Here we discuss recent advances in high-throughput technologies for displaying proteins on functional protein microarrays and the real-time label-free detection of interactions using probes of the local index of refraction, carbon nanotubes and nanowires, or microelectromechanical systems cantilevers. The combination of these technologies will facilitate the large-scale study of protein interactions with proteins as well as with other biomolecules.     

Advances in encapsulin nanocompartment biology and engineering

Compartmentalization is an essential feature of all cells. It allows cells to segregate and coordinate physiological functions in a controlled and ordered manner. Different mechanisms of compartmentalization exist, with the most relevant to prokaryotes being encapsulation via self‐assembling protein‐based compartments. One widespread example of such is that of encapsulins—cage‐like protein nanocompartments able to compartmentalize specific reactions, pathways, and processes in bacteria and archaea. While still relatively nascent bioengineering tools, encapsulins exhibit many promising characteristics, including a number of defined compartment sizes ranging from 24 to 42 nm, straightforward expression, the ability to self‐assemble via the Hong Kong 97‐like fold, marked physical robustness, and internal and external handles primed for rational genetic and molecular manipulation. Moreover, encapsulins allow for facile and specific encapsulation of native or heterologous cargo proteins via naturally or rationally fused targeting peptide sequences. Taken together, the attributes of encapsulins promise substantial customizability and broad usability. This review discusses recent advances in employing engineered encapsulins across various fields, from their use as bionanoreactors to targeted delivery systems and beyond. A special focus will be provided on the rational engineering of encapsulin systems and their potential promise as biomolecular research tools.     

High quality NMR structures: a new force field with implicit water and membrane solvation for Xplor-NIH

Structure determination of proteins by NMR is unique in its ability to measure restraints, very accurately, in environments and under conditions that closely mimic those encountered in vivo. For example, advances in solid-state NMR methods enable structure determination of membrane proteins in detergent-free lipid bilayers, and of large soluble proteins prepared by sedimentation, while parallel advances in solution NMR methods and optimization of detergent-free lipid nanodiscs are rapidly pushing the envelope of the size limit for both soluble and membrane proteins. These experimental advantages, however, are partially squandered during structure calculation, because the commonly used force fields are purely repulsive and neglect solvation, Van der Waals forces and electrostatic energy. Here we describe a new force field, and updated energy functions, for protein structure calculations with EEFx implicit solvation, electrostatics, and Van der Waals Lennard-Jones forces, in the widely used program Xplor-NIH. The new force field is based primarily on CHARMM22, facilitating calculations with a wider range of biomolecules. The new EEFx energy function has been rewritten to enable OpenMP parallelism, and optimized to enhance computation efficiency. It implements solvation, electrostatics, and Van der Waals energy terms together, thus ensuring more consistent and efficient computation of the complete nonbonded energy lists. Updates in the related python module allow detailed analysis of the interaction energies and associated parameters. The new force field and energy function work with both soluble proteins and membrane proteins, including those with cofactors or engineered tags, and are very effective in situations where there are sparse experimental restraints. Results obtained for NMR-restrained calculations with a set of five soluble proteins and five membrane proteins show that structures calculated with EEFx have significant improvements in accuracy, precision, and conformation, and that structure refinement can be obtained by short relaxation with EEFx to obtain improvements in these key metrics. These developments broaden the range of biomolecular structures that can be calculated with high fidelity from NMR restraints.     

Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy

Atomic force microscopy (AFM) has been applied in many biological investigations in the past 15 years. This review focuses on the application of AFM for quantitatively characterizing the structural and thermodynamic properties of protein-protein and protein-nucleic acid complexes. AFM can be used to determine the stoichiometries and association constants of multiprotein assemblies and to quantify changes in conformations of proteins and protein-nucleic acid complexes. In addition, AFM in solution permits the observation of the dynamic properties of biomolecular complexes and the measurement of intermolecular forces between biomolecules. Recent advances in cryogenic AFM, AFM on two-dimensional crystals, carbon nanotube probes, solution imaging, high-speed AFM, and manipulation capabilities enhance these applications by improving AFM resolution and the dynamic and operative capabilities of the AFM. These developments make AFM a powerful tool for investigating the biomolecular assemblies and interactions that govern gene regulation.     

Protein engineering for metabolic engineering: Current and next-generation tools

Protein engineering in the context of metabolic engineering is increasingly important to the field of industrial biotechnology. As the demand for biologically produced food, fuels, chemicals, food additives, and pharmaceuticals continues to grow, the ability to design and modify proteins to accomplish new functions will be required to meet the high productivity demands for the metabolism of engineered organisms. We review advances in selecting, modeling, and engineering proteins to improve or alter their activity. Some of the methods have only recently been developed for general use and are just beginning to find greater application in the metabolic engineering community. We also discuss methods of generating random and targeted diversity in proteins to generate mutant libraries for analysis. Recent uses of these techniques to alter cofactor use; produce non-natural amino acids, alcohols, and carboxylic acids; and alter organism phenotypes are presented and discussed as examples of the successful engineering of proteins for metabolic engineering purposes.     

Use of proteomics for the identification of novel drug targets in brain diseases

In spite of the rapid advances in the development of the new proteomic technologies, there are, to date, relatively fewer studies aiming to explore the neuronal proteome. One of the reasons is the complexity of the brain, which presents high cellular heterogeneity and a unique subcellular compartmentalization. Therefore, tissue fractionation of the brain to enrich proteins of interest will reduce the complexity of the proteomics approach leading to the production of manageable and meaningful results. In this review, general considerations and strategies of proteomics, the advantages and challenges to exploring the neuronal proteome are described and summarized. In addition, this article presents an overview of recent advances of proteomic technologies and shows that proteomics can serve as a valuable tool to globally explore the changes in brain proteome during various disease states. Understanding the molecular basis of brain function will be extremely useful in identifying novel targets for the treatment of brain diseases.     

Advances, challenges, and limitations in serum-proteome-based cancer diagnosis

Recent advances in medicine have dramatically reduced the incidence and mortality of many cardiovascular, infectious, and certain neoplastic diseases; the overall mortality for most malignant solid tumors remains high. The poor prognosis in these cancers is due, in part, to the absence of adequate early screening tests, leading to delays in diagnosis. Three strategies have been applied to fight cancer: analysis of the molecular mechanisms involved in its pathogenesis and progression, improvement of early diagnosis, and the development of novel treatment strategies. There have been major advances in our understanding of cancer biology and pathogenesis and in the development of new (targeted) treatment modalities. However, insufficient progress has been made with respect to improving the methods for the early diagnosis and screening of many cancers. Therefore, cancer is often diagnosed at advanced stages, delaying timely treatment and leading to poor prognosis. Proteome analysis has recently been used for the identification of biomarkers or biomarker patterns that may allow for the early diagnosis of cancer. This tool is of special interest, since it allows for the identification of tumor-derived secretory products in serum or other body fluids. In addition, it may be used to detect reduced levels or loss of proteins in the serum of cancer patients that are present in noncancer individuals. These changes in the serum proteome may result from cancer-specific metabolic or immunological alterations, which are, at least partly, independent of tumor size or mass, thereby facilitating early discovery.     

High-throughput screening for high-efficiency small-molecule biosynthesis

Systems metabolic engineering faces the formidable task of rewiring microbial metabolism to cost-effectively generate high-value molecules from a variety of inexpensive feedstocks for many different applications. Because these cellular systems are still too complex to model accurately, vast collections of engineered organism variants must be systematically created and evaluated through an enormous trial-and-error process in order to identify a manufacturing-ready strain. The high-throughput screening of strains to optimize their scalable manufacturing potential requires execution of many carefully controlled, parallel, miniature fermentations, followed by high-precision analysis of the resulting complex mixtures. This review discusses strategies for the design of high-throughput, small-scale fermentation models to predict improved strain performance at large commercial scale. Established and promising approaches from industrial and academic groups are presented for both cell culture and analysis, with primary focus on microplate- and microfluidics-based screening systems.     

Residual dipolar couplings: Synergy between NMR and structural genomics

Structural genomics is on a quest for the structure and function of a significant fraction of gene products. Current efforts are focusing on structure determination of single-domain proteins, which can readily be targeted by X-ray crystallography, NMR spectroscopy and computational homology modeling. However, comprehensive association of gene products with functions also requires systematic determination of more complex protein structures and other biomolecules participating in cellular processes such as nucleic acids, and characterization of biomolecular interactions and dynamics relevant to function. Such NMR investigations are becoming more feasible, not only due to recent advances in NMR methodology, but also because structural genomics is providing valuable structural information and new experimental and computational tools. The measurement of residual dipolar couplings in partially oriented systems and other new NMR methods will play an important role in this synergistic relationship between NMR and structural genomics. Both an expansion in the domain of NMR application, and important contributions to future structural genomics efforts can be anticipated.     

Protein and RNA engineering to customize microbial molecular reporting

Nature takes advantage of the malleability of protein and RNA sequence and structure to employ these macromolecules as molecular reporters whose conformation and functional roles depend on the presence of a specific ligand (an "effector" molecule). By following nature's example, ligand-responsive proteins and RNA molecules are now routinely engineered and incorporated into customized molecular reporting systems (biosensors). Microbial small-molecule biosensors and endogenous molecular reporters based on these sensing components find a variety of applications that include high-throughput screening of biosynthesis libraries, environmental monitoring, and novel gene regulation in synthetic biology. Here, we review recent advances in engineering small-molecule recognition by proteins and RNA and in coupling in vivo ligand binding to reporter-gene expression or to allosteric activation of a protein conferring a detectable phenotype. Emphasis is placed on microbial screening systems that serve as molecular reporters and facilitate engineering the ligand-binding component to recognize new molecules.     

Manufacture of biopharmaceutical proteins by mammalian cell culture systems

In the last several years, dramatic advances have been in the development of new biopharmaceuticals including monoclonal antibodies for diagnosis and treatment and such genetically engineered proteins as tPA, Factor VIIIc, erythropoietin and soluble CD4, an anti-AIDS protein. Currently, there are several hundred such candidate drugs in human clinical trials. In most cases, these protein-based drugs will require manufacture by mammalian cell culture due to the inability of lower organisms to properly glycosylate, fold, make correct disulfide bonds and secrete active biomolecular forms. The need for large scale production from cell culture will greatly increase as more of the products in clinical trials are approved for commercial production. This will require significant reduction in manufacturing costs per gram, concomitant with increased capacity to hundreds or perhaps even thousands of kilograms annually. As an example, Invitron's multi-reactor manufacturing facility has operated at greater than one-half million liters per year and has experience with more than 250 mammalian cell lines for producing protein drug products.