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The ability to design efficient enzymes from scratch would have a profound effect on chemistry, biotechnology and medicine. Rapid progress in protein engineering over the past decade makes us optimistic that this ambition is within reach. The development of artificial enzymes containing metal cofactors and noncanonical organocatalytic groups shows how protein structure can be optimized to harness the reactivity of nonproteinogenic elements. In parallel, computational methods have been used to design protein catalysts for diverse reactions on the basis of fundamental principles of transition state stabilization. Although the activities of designed catalysts have been quite low, extensive laboratory evolution has been used to generate efficient enzymes. Structural analysis of these systems has revealed the high degree of precision that will be needed to design catalysts with greater activity. To this end, emerging protein design methods, including deep learning, hold particular promise for improving model accuracy. Here we take stock of key developments in the field and highlight new opportunities for innovation that should allow us to transition beyond the current state of the art and enable the robust design of biocatalysts to address societal needs., (© 2022. Springer Nature Limited.)

The success of protein engineering and design has extensively expanded the protein space, which presents a promising strategy for creating next-generation proteins of diverse functions. Among these proteins, the synthetic binding proteins (SBPs) are smaller, more stable, less immunogenic, and better of tissue penetration than others, which make the SBP-related data attracting extensive interest from worldwide scientists. However, no database has been developed to systematically provide the valuable information of SBPs yet. In this study, a database named 'Synthetic Binding Proteins for Research, Diagnosis, and Therapy (SYNBIP)' was thus introduced. This database is unique in (a) comprehensively describing thousands of SBPs from the perspectives of scaffolds, biophysical & functional properties, etc.; (b) panoramically illustrating the binding targets & the broad application of each SBP and (c) enabling a similarity search against the sequences of all SBPs and their binding targets. Since SBP is a human-made protein that has not been found in nature, the discovery of novel SBPs relied heavily on experimental protein engineering and could be greatly facilitated by in-silico studies (such as AI and computational modeling). Thus, the data provided in SYNBIP could lay a solid foundation for the future development of novel SBPs. The SYNBIP is accessible without login requirement at both official (https://idrblab.org/synbip/) and mirror (http://synbip.idrblab.net/) sites., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to disclose.

Linkers play critical roles in the construction of synthetic protein switches and sensors as they functionally couple a receptor with an actuator. With an increasing number of molecular toolboxes and experimental strategies becoming available that can be applied to engineer protein switches and sensors with tailored response functions, optimising the connecting linkers remains an idiosyncratic and empiric process. This review aims to provide an in-depth analysis of linker motifs, the biophysical properties they confer, and how they impact the performance of synthetic protein switches and sensors while identifying trends, mechanisms, and strategies that underlie the most potent switches and sensors., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The discovery of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, and its development into a set of powerful tools for manipulating the genome, has revolutionized genome editing. Precise, targeted CRISPR/Cas-based genome editing has become the most widely used platform in organisms ranging from plants to animals. The CRISPR/Cas system has been extensively modified to increase its efficiency and fidelity. In addition, the fusion of various protein motifs to Cas effector proteins has facilitated diverse set of genetic manipulations, such as base editing, transposition, recombination, and epigenetic regulation. The CRISPR/Cas system is undergoing continuous development to overcome current limitations, including off-target effects, narrow targeting scope, and issues associated with the delivery of CRISPR components for genome engineering and therapeutic approaches. Here, we review recent progress in a diverse array of CRISPR/Cas-based tools. We also describe limitations and concerns related to the use of CRISPR/Cas technologies.

Enzyme engineering is an indispensable tool in the field of synthetic biology, where enzymes are challenged to carry out novel or improved functions. Achieving these goals sometimes goes beyond modifying the primary sequence of the enzyme itself. The use of protein or nucleic acid scaffolds to enhance enzyme properties has been reported for applications such as microbial production of chemicals, biosensor development and bioremediation. Key advantages of using these assemblies include optimizing reaction conditions, improving metabolic flux and increasing enzyme stability. This review summarizes recent trends in utilizing genetically encodable scaffolds, developed in line with synthetic biology methodologies, to complement the purposeful deployment of enzymes. Current molecular tools for constructing these synthetic enzyme-scaffold systems are also highlighted.

CRISPR technology is a universal tool for genome engineering that has revolutionized biotechnology. Recently identified unique CRISPR/Cas systems, as well as re-engineered Cas proteins, have rapidly expanded the functions and applications of CRISPR/Cas systems. The structures of Cas proteins are complex, containing multiple functional domains. These protein domains are evolutionarily conserved polypeptide units that generally show independent structural or functional properties. In this review, we propose using protein domains as a new way to classify protein engineering strategies for these proteins and discuss common ways to engineer key domains to modify the functions of CRISPR/Cas systems., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The hydrolysis of mannans by endo-β-mannanases continues to gather significance as exemplified by its commercial applications in food, feed, and a rekindled interest in biorefineries. The present review provides a comprehensive account of fundamental research and fascinating insights in the field of endo-β-mannanase engineering in order to improve over-expression and to decipher molecular determinants governing activity-stability during harsh conditions, substrate recognition, polysaccharide specificity, endo/exo mode of action and multi-functional activities in the modular polypeptide. In-depth analysis of the available literature has also been made on rational and directed evolution approaches, which have translated native endo-β-mannanases into superior biocatalysts for satisfying industrial requirements.

Tremendous advancements in cell and protein engineering methodologies and bioinformatics have led to a vast increase in bacterial production clones and recombinant protein variants to be screened and evaluated. Consequently, an urgent need exists for efficient high-throughput (HTP) screening approaches to improve the efficiency in early process development as a basis to speed-up all subsequent steps in the course of process design and engineering. In this study, we selected the BioLector micro-bioreactor (µ-bioreactor) system as an HTP cultivation platform to screen E. coli expression clones producing representative protein candidates for biopharmaceutical applications. We evaluated the extent to which generated clones and condition screening results were transferable and comparable to results from fully controlled bioreactor systems operated in fed-batch mode at moderate or high cell densities. Direct comparison of 22 different production clones showed great transferability. We observed the same growth and expression characteristics, and identical clone rankings except one host-Fab-leader combination. This outcome demonstrates the explanatory power of HTP µ-bioreactor data and the suitability of this platform as a screening tool in upstream development of microbial systems. Fast, reliable, and transferable screening data significantly reduce experiments in fully controlled bioreactor systems and accelerate process development at lower cost.

The rise of antibodies as a promising and rapidly growing class of biotherapeutic proteins has motivated numerous studies to characterize and understand antibody structures. In the past decades, the number of antibody crystal structures increased substantially, which revolutionized the atomistic understanding of antibody functions. Even though numerous static structures are known, various biophysical properties of antibodies (i.e., specificity, hydrophobicity and stability) are governed by their dynamic character. Additionally, the importance of high-quality structures in structure-function relationship studies has substantially increased. These structure-function relationship studies have also created a demand for precise homology models of antibody structures, which allow rational antibody design and engineering when no crystal structure is available. Here, we discuss various aspects and challenges in antibody design and extend the paradigm of describing antibodies with only a single static structure to characterizing them as dynamic ensembles in solution.

In recent years, extensive attention has been given to the generation of new classes of ligand- specific binding proteins to supplement monoclonal antibodies. A combination of protein engineering and display technologies has been used to manipulate non-human antibodies for humanization and stabilization purposes or even the generation of new binding proteins. Engineered protein scaffolds can now be directed against therapeutic targets to treat cancer and immunological disorders. Although very few of these scaffolds have successfully passed clinical trials, their remarkable properties such as robust folding, high solubility, and small size motivate their employment as a tool for biology and applied science studies. Here, we have focused on the generation of new non-Ig binding proteins and single domain antibody manipulation, with a glimpse of their applications., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Production of biologics in plants, or plant molecular pharming, is a promising protein expression technology that is receiving increasing attention from the pharmaceutical industry. Previously, low expression yields of recombinant proteins and the realization that certain post-translational modifications (PTMs) may not occur optimally limited the widespread acceptance of the technology. However, molecular engineering of the plant secretory pathway is now enabling the production of increasingly complex biomolecules using tailored protein-specific approaches to ensure their maturation. These involve the elimination of undesired processing events, and the introduction of heterologous biosynthetic machinery to support the production of specific target proteins. Here, we discuss recent advances in the production of pharmaceutical proteins in plants, which leverage the unique advantages of the technology., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Natural evolution has generated an impressively diverse protein universe via duplication and recombination from a set of protein fragments that served as building blocks. The application of these concepts to the design of new proteins using subdomain-sized fragments from different folds has proven to be experimentally successful. To better understand how evolution has shaped our protein universe, we performed an all-against-all comparison of protein domains representing all naturally existing folds and identified conserved homologous protein fragments. Overall, we found more than 1000 protein fragments of various lengths among different folds through similarity network analysis. These fragments are present in very different protein environments and represent versatile building blocks for protein design. These data are available in our web server called F(old P)uzzle (fuzzle.uni-bayreuth.de), which allows to individually filter the dataset and create customized networks for folds of interest. We believe that our results serve as an invaluable resource for structural and evolutionary biologists and as raw material for the design of custom-made proteins., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Thioredoxins are a family of conserved oxidoreductases responsible for maintaining redox balance within cells. They have also served as excellent model systems for protein design and engineering studies particularly through ancestral sequence reconstruction methods. The recent work by Gamiz-Arco et al. [Biochem J (2019) 476, 3631-3647] answers fundamental questions on how specific sequence differences can contribute to differences in folding rates between modern and ancient thioredoxins but also among a selected subset of modern thioredoxins. They surprisingly find that rapid unassisted folding, a feature of ancient thioredoxins, is not conserved in the modern descendants suggestive of co-evolution of better folding machinery that likely enabled the accumulation of mutations that slow-down folding. The work thus provides an interesting take on the expected folding-stability-function constraint while arguing for additional factors that contribute to sequence evolution and hence impact folding efficiency., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Two decades into the twenty-first century, a confluence of breakthrough technologies wielded at the molecular level is presenting biologists with unique opportunities to unravel the complexities of the cellular world. CRISPR/Cas9 allows gene knock-outs, knock-ins, and single-base editing at chromosomal loci. RNA-based tools such as siRNA, antisense oligos, and morpholinos can be used to silence expression of specific genes. Meanwhile, protein knockdown tools that draw inspiration from natural regulatory mechanisms and facilitate elimination of native or degron-tagged proteins from cells are rapidly emerging. The acute and reversible reduction in protein levels enabled by these methods allows for precise determination of loss-of-function phenotypes free from secondary effects or compensatory adaptation that can confound nucleic-acid-based methods that involve slow depletion or permanent loss of a protein. In this Review, we summarize the ingenious ways biologists have exploited natural mechanisms for protein degradation to direct the elimination of specific proteins at will. This has led to advancements not only in basic research but also in the therapeutic space with the introduction of PROTACs into clinical trials for cancer patients., Competing Interests: Declaration of Interests R.V., D.M., and R.J.D. are employees and stock holders of AMGEN. R.J.D. is additionally SVP of AMGEN Global Research., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Most proteins are specifically localized in membrane-encapsulated organelles or non-membrane-bound compartments. The subcellular localization of proteins facilitates their functions and integration into functional networks; therefore, protein localization is tightly regulated in diverse biological contexts. However, protein localization has been mainly analyzed through immunohistochemistry or the fractionation of subcellular compartments, each of which has major drawbacks. Immunohistochemistry can examine only a handful of proteins at a time, and fractionation inevitably relies on the lysis of cells, which disrupts native cellular conditions. Recently, an engineered ascorbate peroxidase (APEX)-based proximity labeling technique combined with mass spectrometry was developed, which allows for temporally and spatially resolved proteomic mapping. In the presence of H 2 O 2 , engineered APEX oxidizes biotin-phenols into biotin-phenoxyl radicals, and these short-lived radicals biotinylate electron-rich amino acids within a radius of several nanometers. Biotinylated proteins are subsequently enriched by streptavidin and identified by mass spectrometry. This permits the sensitive and efficient labeling of proximal proteins around locally expressed APEX. Through the targeted expression of APEX in the subcellular region of interest, proteomic profiling of submitochondrial spaces, the outer mitochondrial membrane, the endoplasmic reticulum (ER)-mitochondrial contact, and the ER membrane has been performed. Furthermore, this method has been modified to define interaction networks in the vicinity of target proteins and has also been applied to analyze the spatial transcriptome. In this Perspective, we provide an outline of this newly developed technique and discuss its potential applications to address diverse biological questions.

Engineering of nucleic acids has been a goal in research for many years. Since the discovery of catalytic nucleic acids (ribozymes and DNAzymes), this field has attracted even more attention. One reason for the increased interest is that a large number of ribozymes have been engineered that catalyze a broad range of reactions of relevance to the origin of life. Another reason is that the structures of ribozymes or DNAzymes have been modulated such that activity is dependent on allosteric regulation by an external cofactor. Such constructs have great potential for application as biosensors in medicinal or environmental diagnostics, and as molecular tools for control of cellular processes. In addition to the development of nucleic acid enzymes by in vitro selection, rational design is a powerful strategy for the engineering of ribozymes or DNAzymes with tailored features. The structures and mechanisms of a large number of nucleic acid catalysts are now well understood. Therefore, specific design of their functional properties by structural modulation is a good option for the development of custom-made molecular tools. For rational design, several parameters have to be considered, and a number of tools are available to help/guide sequence design. Here, we discuss sequence, structural and functional design using the example of hairpin ribozyme variants to highlight the challenges and opportunities of rational nucleic enzyme engineering.

Clustered regularly interspaced short palindromic repeats (CRISPR) form the adaptive immune system in archaea and bacteria and have been modified for genome engineering in eukaryotic cells. CRISPR systems contain 2 components, a single-guide RNA, which is a short RNA composed of a 20 nucleotide sequence that targets specific sites in the genomic DNA and a scaffold necessary for its binding to the CRISPR-associated endonuclease (Cas9). Because of its high efficiency and accuracy, the CRISPR-Cas9 genome editing based therapies are poised to treat a multitude of human diseases with a promise to target previously "undruggable" proteins. As the first in-body clinical trial with CRISPR-Cas9 is embarked on, the risks associated with administering the genome editing machinery to patients become increasingly relevant. Recent studies have demonstrated an innate and adaptive cellular immune response to Cas9 in mouse models and the presence of anti-Cas9 antibodies and T-cells in human plasma. Pre-existing immunity against therapeutic Cas9 delivery could decrease its efficacy in vivo and may pose significant safety issues. This review focuses on the immunogenicity of the Cas9 protein and summarizes potential approaches to circumvent the problem of immune recognition., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Computational design of binding sites in proteins remains difficult, in part due to limitations in our current ability to sample backbone conformations that enable precise and accurate geometric positioning of side chains during sequence design. Here we present a benchmark framework for comparison between flexible-backbone design methods applied to binding interactions. We quantify the ability of different flexible backbone design methods in the widely used protein design software Rosetta to recapitulate observed protein sequence profiles assumed to represent functional protein/protein and protein/small molecule binding interactions. The CoupledMoves method, which combines backbone flexibility and sequence exploration into a single acceptance step during the sampling trajectory, better recapitulates observed sequence profiles than the BackrubEnsemble and FastDesign methods, which separate backbone flexibility and sequence design into separate acceptance steps during the sampling trajectory. Flexible-backbone design with the CoupledMoves method is a powerful strategy for reducing sequence space to generate targeted libraries for experimental screening and selection., (© 2019 Wiley Periodicals, Inc.)

Cytokines and biological toxins represent two potent classes of biomolecules that have long been explored for their potential as therapeutics. Considerable side effects and poor pharmacokinetics frequently observed with both have limited their broad application. Recombinant protein engineering has allowed the construction of immunocytokines and immunotoxins that seek to exploit the advantageous properties of immunoglobulins to address these issues. Whole antibodies, antibody fragments, constant domains and derivatives have been fused genetically to a range of cytokines and toxins. This review considers the strategies that have been employed and the problems sought to be resolved in the clinical evaluation of this class of biotherapeutic., (© 2019 médecine/sciences – Inserm.)

Over the past ten years, an increased knowledge of tumor biology and immunology allowed the design and development of novel therapeutic antibody and protein scaffold formats, where bispecific antibodies (Abs) play a major role. The latter molecules can (1) bring novel pharmacological properties through the co-engagement of two targets, (2) increase the safety profile as compared to a combination of two antibodies thanks to a targeted relocation to the tumor and (3) reduce development and manufacturing costs associated with single drug product. This review analyzes the different bispecific antibodies and scaffolds described in the field of immuno-oncology, their structure and major pharmacological and physico-chemical properties., (© 2019 médecine/sciences – Inserm.)

The hinge region is a short sequence of the heavy chains (H) of antibodies linking the Fab (Fragment antigen binding) region to the Fc (Fragment crystallisable) region. The functional properties of the four IgG subclasses partly result from the sequence differences of their hinge regions as some amino acids of the lower hinge region are located within or in the close vicinity of the C1q and FcγR binding sites on the IgG H chains. In addition, the hinge is susceptible to proteolytic cleavage by many proteases present in tumor and/or inflammatory microenvironment capable of affecting functional responses. Thus, an optimal format of the hinge region remains a major challenge for the development of new therapeutic antibodies., (© 2019 médecine/sciences – Inserm.)

Introduction : The practice of turning scientific data into music, a practice known as sonification, is a growing field. Driven by analogies between the hierarchical structures of proteins and many forms of music, multiple attempts of mapping proteins to music have been made. Previous works have either worked at a low level, mapping amino acid to notes, or at a higher level, using the overall structure as a basis for composition. Areas covered : We report a comprehensive mapping strategy that encompasses the encoding of the geometry of proteins, in addition to the amino acid sequence and secondary structure information. This leads to a piece of music that is both more complete and closely linked to the original protein. By using this mapping, we can invert the process and map music to proteins, retrieving not only the amino acid sequence but also the secondary structure and folding from musical data. Expert opinion : We can train a machine learning model on 'protein music' to generate new music that can be translated to new proteins. By selecting proper datasets and conditioning parameters on the generative model, we could tune de novo proteins with high level parameters to achieve certain protein design features.

With the advancement of medicine, the utility of protein therapeutics is increasing exponentially. However, a significant number of protein therapeutics suffer from grave limitations, which include their subpar pharmacokinetics. In this study, we have reviewed the emerging field of protein chimerization for improving the short circulatory half-life of protein therapeutics. We have discussed various aspects of protein therapeutics aiming at their mechanism of clearance and various approaches used to increase their short circulatory half-life with principal focus on the concept of chimerization. Furthermore, we have comprehensively reviewed various components of chimera, such as half-life extension partners and linkers, their shortcomings, and prospective work to be undertaken for developing effective chimeric protein therapeutics., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Cytochromes P450 (P450 or CYP) are heme-containing enzymes that catalyze the introduction of one atom of molecular oxygen into nonactivated C-H bonds, often in a regio- and stereoselective manner. This ability, combined with a tremendous number of accepted substrates, makes P450s powerful biocatalysts. Sixty years after their discovery, P450 systems are recognized as essential bio-bricks in synthetic biology approaches to enable production of high-value complex molecules in recombinant hosts. Recent impressive results in protein engineering led to P450s with tailored properties that are even able to catalyze abiotic reactions. The introduction of P450s in artificial multi-enzymatic cascades reactions and chemo-enzymatic processes offers exciting future perspectives to access novel compounds that cannot be synthesized by nature or by chemical routes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The hairpin ribozyme is a small, naturally occurring RNA that catalyzes the reversible cleavage of RNA substrates. Among the small endonucleolytic ribozymes, the hairpin ribozyme possesses the unique feature of the internal equilibrium between cleavage and ligation being shifted toward ligation. This allows control of the reaction outcome by structural design: fragments that are strongly bound to the ribozyme are preferentially ligated, whereas substrates that easily dissociate upon cleavage, such that they are not available for religation, are preferentially cleaved. We have made use of this characteristic feature in engineering a number of hairpin ribozyme variants by programmed conformational design that carry out cascades of cleavage and ligation reactions, and as a result mediate more complex RNA processing reactions. Here, we review our work on the engineering of hairpin ribozyme variants for RNA recombination and regular and back-splicing, and discuss the relevance of such activities in early life., (© 2019 New York Academy of Sciences.)

Enzymes generated by natural recruitment and protein engineering have greatly contribute in various sets of applications. However, their insufficient stability is a bottleneck that limit the rapid development of biocatalysis. Novel approaches based on precise and global structural dissection, advanced gene manipulation, and combination with the multidisciplinary techniques open a new horizon to generate stable enzymes efficiently. Here, we comprehensively introduced emerging advances of protein engineering strategies for enzyme stabilization. Then, we highlighted practical cases to show importance of enzyme stabilization in pharmaceutical and industrial applications. Combining computational enzyme design with molecular evolution will hold considerable promise in this field., (Copyright © 2018. Published by Elsevier Inc.)

With the rapid development of DNA synthesis and next-generation sequencing, synthetic biology that aims to standardize, modularize, and innovate cellular functions, has achieved vast progress. Here we review key advances in synthetic biology of the yeast Saccharomyces cerevisiae, which serves as an important eukaryal model organism and widely applied cell factory. This covers the development of new building blocks, i.e., promoters, terminators and enzymes, pathway engineering, tools developments, and gene circuits utilization. We will also summarize impacts of synthetic biology on both basic and applied biology, and end with further directions for advancing synthetic biology in yeast.

Alarming changes in environmental conditions have prompted significant research into producing renewable commodities from sources other than fossil fuels. One such alternative is CO 2 , a determinate greenhouse gas with historically high atmospheric levels. If sequestered, CO 2 could be used as a highly renewable feedstock for industrially relevant products and fuels. The vast majority of atmospheric CO 2 fixation is accomplished by photosynthetic organisms, which have unfortunately proven difficult to utilize as chassis for industrial production. Nonphotosynthetic CO 2 fixing microorganisms and pathways have recently attracted scientific and commercial interest. This Perspective will review promising alternate CO 2 fixation strategies and their potential to supply microbially produced fuels and commodity chemicals, such as higher alcohols. Acetogenic fermentation and microbial electrosynthesis are the primary focuses of this review.

Synthetic biologists have developed sophisticated molecular and genetic tools to engineer new biochemical functions in cells. Applications for these tools have focused on important problems in energy and medicine, but they can also be applied to address basic science topics that cannot be easily accessed by classical approaches. We focus on recent work that has utilized synthetic biology approaches, ranging from promoter engineering to the de novo synthesis of cellular parts, to investigate a wide range of biochemical and cellular questions. Insights obtained by these efforts include how fatty acid composition mediates cellular metabolism, how transcriptional circuits act to stabilize multicellular networks, and fitness trade-offs involved in the selection of genetic regulatory elements. We also highlight common themes about how "discovery by synthesis" approaches can aid fundamental research. For example, rewiring of native metabolism through metabolic engineering is a powerful tool for investigating biological molecules whose exact composition and abundance are key for function. Meanwhile, endeavors to synthesize cells and their components allow scientists to address evolutionary questions that are otherwise constrained by extant laboratory models.

The reductionist approach is prevalent in biomedical science. However, increasing evidence now shows that biological systems cannot be simply considered as the sum of its parts. With experimental, technological, and computational advances, we can now do more than view parts in isolation, thus we propose that an increasing holistic view (where a protein is investigated as much as a whole as possible) is now timely. To further advocate this, we review and discuss several studies and applications involving allostery, where distant protein regions can cross-talk to influence functionality. Therefore, we believe that an increasing big picture approach holds great promise, particularly in the areas of antibody engineering and drug discovery in rational drug design., (© 2019 The Author(s).)

Our ability to engineer protein structure and function has grown dramatically over recent years. Perhaps the next level in protein design is to develop proteins whose function can be regulated in response to various stimuli, including ligand binding, pH changes, and light. Endeavors toward these goals have tested and expanded on our understanding of protein function and allosteric regulation. In this chapter, we provide examples from different methods for developing new allosterically regulated proteins. These methods range from whole insertion of regulatory domains into new host proteins, to covalent attachment of photoswitches to generate light-responsive proteins, and to targeted changes to specific amino acid residues, especially to residues identified to be important for relaying allosteric information across the protein framework. Many of the examples we discuss have already found practical use in medical and biotechnology applications.

This year, the Nobel Prize in Chemistry was awarded to three pioneering scientists who applied laboratory evolution for protein engineering: Frances Arnold, George P. Smith, and Sir Gregory P. Winter. This approach has had major impact in various applications and inspires the search for the general principles of design through evolution., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In the period 1985 to 1995 applications of biocatalysis, driven by the need for more sustainable manufacture of chemicals and catalytic, (enantio)selective methods for the synthesis of pharmaceutical intermediates, largely involved the available hydrolases. This was followed, in the next two decades, by revolutionary developments in protein engineering and directed evolution for the optimisation of enzyme function and performance that totally changed the biocatalysis landscape. In the same period, metabolic engineering and synthetic biology revolutionised the use of whole cell biocatalysis in the synthesis of commodity chemicals by fermentation. In particular, developments in the enzymatic enantioselective synthesis of chiral alcohols and amines are highlighted. Progress in enzyme immobilisation facilitated applications under harsh industrial conditions, such as in organic solvents. The emergence of biocatalytic or chemoenzymatic cascade processes, often with co-immobilised enzymes, has enabled telescoping of multi-step processes. Discovering and inventing new biocatalytic processes, based on (meta)genomic sequencing, evolving enzyme promiscuity, chemomimetic biocatalysis, artificial metalloenzymes, and the introduction of non-canonical amino acids into proteins, are pushing back the limits of biocatalysis function. Finally, the integral role of biocatalysis in developing a biobased carbon-neutral economy is discussed.

As a key parameter impacting functional and structural heterogeneity, protein glycosylation is a critical quality attribute for antibody biotherapeutic manufacturing. The glycan patterns on recombinant antibodies, particularly on the conserved fragment crystallizable (Fc) region, can have significant effects on an antibody's functional activities including clearance rate, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and anti-inflammatory activity. In this review, we examined specific glycan attachments (fucosylation, sialylation, galactosylation, high-mannose, and bisecting glycans) and their importance to antibody properties. Next, we summarized the recent and current achievements on controlling antibody glycoforms in Chinese hamster ovary (CHO) and other mammalian cells through multiple strategies including genetic engineering, protein engineering, media modification, and other emerging technologies. Further, the impact of one carbohydrate modification on other glycan structures is also described. Finally, approaches to generate desirable homogenous glycan profiles on antibodies are also detailed. By applying multiple complementary intracellular and extracellular strategies, biotechnologists are well on their ways to precisely tuning antibody glycoforms emerging from bioreactors in the coming decades., (© 2018 Wiley Periodicals, Inc.)

Some of the recombinant protein therapeutics with short half-life requires high frequent dose or injection, which results in poor patient compliance. This challenge has prompted the development of long-acting recombinant proteins in recent years. Four strategies and methods, including chemical modification, protein engineering, fusion proteins and protein glycosylation are used to modify protein molecule and finally obtain improved pharmacokinetics (PK) properties. This article reviews the four strategies of half-life extension and presents a detailed list of long-acting therapeutics on US, EU and China markets.

Applied biocatalysis is driven by environmental and economic incentives for using enzymes in the synthesis of various pharmaceutical and industrially important chemicals. Protein engineering is used to tailor the properties of enzymes to catalyze desired chemical transformations, and some engineered enzymes now outperform the best chemocatalytic alternatives by orders of magnitude. Unfortunately, custom engineering of a robust biocatalyst is still a time-consuming process, but an understanding of how enzyme function depends on amino acid sequence will speed up the process. This review demonstrates how recent advances in ultrahigh-throughput screening, mutational scanning, DNA synthesis, metagenomics, and machine learning will soon make it possible to model, predict, and manipulate the relationship between protein sequence and function, accelerating the tailor design of novel biocatalysts., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Alternative bases that can fit into the DNA double helix have now been used in vivo to direct the synthesis of proteins incorporating unnatural amino acids. This bioengineering feat is significant at both the conceptual and the practical levels., (© 2018 médecine/sciences – Inserm.)

A fundamental goal of protein biochemistry is to determine the sequence-function relationship, but the vastness of sequence space makes comprehensive evaluation of this landscape difficult. However, advances in DNA synthesis and sequencing now allow researchers to assess the functional impact of every single mutation in many proteins, but challenges remain in library construction and the development of general assays applicable to a diverse range of protein functions. This Perspective briefly outlines the technical innovations in DNA manipulation that allow massively parallel protein biochemistry and then summarizes the methods currently available for library construction and the functional assays of protein variants. Areas in need of future innovation are highlighted with a particular focus on assay development and the use of computational analysis with machine learning to effectively traverse the sequence-function landscape. Finally, applications in the fundamentals of protein biochemistry, disease prediction, and protein engineering are presented.

Residing at the interface of chemistry and biotechnology, artificial metalloenzymes (ArMs) offer an attractive technology to combine the versatile reaction repertoire of transition metal catalysts with the exquisite catalytic features of enzymes. While earlier efforts in this field predominantly comprised studies in well-defined test-tube environments, a trend towards exploiting ArMs in more complex environments has recently emerged. Integration of these artificial biocatalysts in enzymatic cascades and using them in whole-cell biotransformations and in vivo opens up entirely novel prospects for both preparative chemistry and synthetic biology. We highlight selected recent developments with a particular focus on challenges and opportunities in the in vivo application of ArMs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

As of May 1, 2017, 74 antibody-based molecules have been approved by a regulatory authority in a major market. Additionally, there are 70 and 575 antibody-based molecules in phase III and phase I/II clinical trials, respectively. These total 719 antibody-based clinical stage molecules include 493 naked IgGs, 87 antibody-drug conjugates, 61 bispecific antibodies, 37 total Fc fusion proteins, 17 radioimmunoglobulins, 13 antibody fragments, and 11 immunocytokines. New uses for these antibodies are being discovered each year. For oncology, many of the exciting new approaches involve antibody modulation of T-cells. There are over 80 antibodies in clinical trials targeting T cell checkpoints, 26 T-cell-redirected bispecific antibodies, and 145 chimeric antigen receptor (CAR) cell-based candidates (all currently in phase I or II clinical trials), totaling more than 250 T cell interacting clinical stage antibody-based candidates. Finally, significant progress has been made recently on routes of delivery, including delivery of proteins across the blood-brain barrier, oral delivery to the gut, delivery to the cellular cytosol, and gene- and viral-based delivery of antibodies. Thus, there are currently at least 864 antibody-based clinical stage molecules or cells, with incredible diversity in how they are constructed and what activities they impart. These are followed by a next wave of novel molecules, approaches, and new methods and routes of delivery, demonstrating that the field of antibody-based biologics is very innovative and diverse in its approaches to fulfill their promise to treat unmet medical needs.

Background: The conservation of the genetic code indicates that there was a single origin, but like all genetic material, the cell's interpretation of the code is subject to evolutionary pressure. Single nucleotide variations in tRNA sequences can modulate codon assignments by altering codon-anticodon pairing or tRNA charging. Either can increase translation errors and even change the code. The frozen accident hypothesis argued that changes to the code would destabilize the proteome and reduce fitness. In studies of model organisms, mistranslation often acts as an adaptive response. These studies reveal evolutionary conserved mechanisms to maintain proteostasis even during high rates of mistranslation., Scope of Review: This review discusses the evolutionary basis of altered genetic codes, how mistranslation is identified, and how deviations to the genetic code are exploited. We revisit early discoveries of genetic code deviations and provide examples of adaptive mistranslation events in nature. Lastly, we highlight innovations in synthetic biology to expand the genetic code., Major Conclusions: The genetic code is still evolving. Mistranslation increases proteomic diversity that enables cells to survive stress conditions or suppress a deleterious allele. Genetic code variants have been identified by genome and metagenome sequence analyses, suppressor genetics, and biochemical characterization., General Significance: Understanding the mechanisms of translation and genetic code deviations enables the design of new codes to produce novel proteins. Engineering the translation machinery and expanding the genetic code to incorporate non-canonical amino acids are valuable tools in synthetic biology that are impacting biomedical research. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Nature uses a limited, conservative set of amino acids to synthesize proteins. The ability to genetically encode an expanded set of building blocks with new chemical and physical properties is transforming the study, manipulation and evolution of proteins, and is enabling diverse applications, including approaches to probe, image and control protein function, and to precisely engineer therapeutics. Underpinning this transformation are strategies to engineer and rewire translation. Emerging strategies aim to reprogram the genetic code so that noncanonical biopolymers can be synthesized and evolved, and to test the limits of our ability to engineer the translational machinery and systematically recode genomes.