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Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins.
Hatlem, D, Trunk, T, Linke, D, Leo, JC
International journal of molecular sciences. 2019;(9)
Abstract
The SpyCatcher-SpyTag system was developed seven years ago as a method for protein ligation. It is based on a modified domain from a Streptococcus pyogenes surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag). Upon recognition, the two form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag. This technology has been used, among other applications, to create covalently stabilized multi-protein complexes, for modular vaccine production, and to label proteins (e.g., for microscopy). The SpyTag system is versatile as the tag is a short, unfolded peptide that can be genetically fused to exposed positions in target proteins; similarly, SpyCatcher can be fused to reporter proteins such as GFP, and to epitope or purification tags. Additionally, an orthogonal system called SnoopTag-SnoopCatcher has been developed from an S. pneumoniae pilin that can be combined with SpyCatcher-SpyTag to produce protein fusions with multiple components. Furthermore, tripartite applications have been produced from both systems allowing the fusion of two peptides by a separate, catalytically active protein unit, SpyLigase or SnoopLigase. Here, we review the current state of the SpyCatcher-SpyTag and related technologies, with a particular emphasis on their use in vaccine development and in determining outer membrane protein localization and topology of surface proteins in bacteria.
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2.
Current Trends in Protein Engineering: Updates and Progress.
Sinha, R, Shukla, P
Current protein & peptide science. 2019;(5):398-407
Abstract
Proteins are one of the most important and resourceful biomolecules that find applications in health, industry, medicine, research, and biotechnology. Given its tremendous relevance, protein engineering has emerged as significant biotechnological intervention in this area. Strategic utilization of protein engineering methods and approaches has enabled better enzymatic properties, better stability, increased catalytic activity and most importantly, interesting and wide range applicability of proteins. In fact, the commercialization of engineered proteins have manifested in economically beneficial and viable solutions for industry and healthcare sector. Protein engineering has also evolved to become a powerful tool contributing significantly to the developments in both synthetic biology and metabolic engineering. The present review revisits the current trends in protein engineering approaches such as rational design, directed evolution, de novo design, computational approaches etc. and encompasses the recent progresses made in this field over the last few years. The review also throws light on advanced or futuristic protein engineering aspects, which are being explored for design and development of novel proteins with improved properties or advanced applications.
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3.
Near-Infrared Fluorescent Proteins and Their Applications.
Karasev, MM, Stepanenko, OV, Rumyantsev, KA, Turoverov, KK, Verkhusha, VV
Biochemistry. Biokhimiia. 2019;(Suppl 1):S32-S50
Abstract
High transparency, low light-scattering, and low autofluorescence of mammalian tissues in the near-infrared (NIR) spectral range (~650-900 nm) open a possibility for in vivo imaging of biological processes at the micro- and macroscales to address basic and applied problems in biology and biomedicine. Recently, probes that absorb and fluoresce in the NIR optical range have been engineered using bacterial phytochromes - natural NIR light-absorbing photoreceptors that regulate metabolism in bacteria. Since the chromophore in all these proteins is biliverdin, a natural product of heme catabolism in mammalian cells, they can be used as genetically encoded fluorescent probes, similarly to GFP-like fluorescent proteins. In this review, we discuss photophysical and biochemical properties of NIR fluorescent proteins, reporters, and biosensors and analyze their characteristics required for expression of these molecules in mammalian cells. Structural features and molecular engineering of NIR fluorescent probes are discussed. Applications of NIR fluorescent proteins and biosensors for studies of molecular processes in cells, as well as for tissue and organ visualization in whole-body imaging in vivo, are described. We specifically focus on the use of NIR fluorescent probes in advanced imaging technologies that combine fluorescence and bioluminescence methods with photoacoustic tomography.
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4.
Synthesis of Human Milk Oligosaccharides: Protein Engineering Strategies for Improved Enzymatic Transglycosylation.
Zeuner, B, Teze, D, Muschiol, J, Meyer, AS
Molecules (Basel, Switzerland). 2019;(11)
Abstract
Human milk oligosaccharides (HMOs) signify a unique group of oligosaccharides in breast milk, which is of major importance for infant health and development. The functional benefits of HMOs create an enormous impetus for biosynthetic production of HMOs for use as additives in infant formula and other products. HMO molecules can be synthesized chemically, via fermentation, and by enzymatic synthesis. This treatise discusses these different techniques, with particular focus on harnessing enzymes for controlled enzymatic synthesis of HMO molecules. In order to foster precise and high-yield enzymatic synthesis, several novel protein engineering approaches have been reported, mainly concerning changing glycoside hydrolases to catalyze relevant transglycosylations. The protein engineering strategies for these enzymes range from rationally modifying specific catalytic residues, over targeted subsite -1 mutations, to unique and novel transplantations of designed peptide sequences near the active site, so-called loop engineering. These strategies have proven useful to foster enhanced transglycosylation to promote different types of HMO synthesis reactions. The rationale of subsite -1 modification, acceptor binding site matching, and loop engineering, including changes that may alter the spatial arrangement of water in the enzyme active site region, may prove useful for novel enzyme-catalyzed carbohydrate design in general.
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5.
Recent Progress in Chemical Modification of Proteins.
Sakamoto, S, Hamachi, I
Analytical sciences : the international journal of the Japan Society for Analytical Chemistry. 2019;(1):5-27
Abstract
Chemical modification of proteins is important for creating a myriad of engineered proteins and for elucidating the function and dynamics of proteins in live cells. A wide variety of chemical protein modification methods have been developed and can be categorized into three classes: (i) modification of proteins using the reactivity of naturally occurring amino acids; (ii) modification by bioorthogonal reactions using unnatural amino acids, most of which can be site-selectively incorporated into proteins-of-interest using genetic codon expansion techniques; and (iii) recognition driven chemical modification, which is the only approach that allows modification of endogenous proteins without any genetic manipulation even under heavily crowded and multi-molecular conditions, as in live cells and organisms. All of these approaches have merits and limitations. In this review, we summarize these approaches and discuss their characteristics with respect to specificity, reaction rate and versatility.
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6.
Evaluating and Enhancing Target Specificity of Gene-Editing Nucleases and Deaminases.
Kim, D, Luk, K, Wolfe, SA, Kim, JS
Annual review of biochemistry. 2019;:191-220
Abstract
Programmable nucleases and deaminases, which include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR RNA-guided nucleases, and RNA-guided base editors, are now widely employed for the targeted modification of genomes in cells and organisms. These gene-editing tools hold tremendous promise for therapeutic applications. Importantly, these nucleases and deaminases may display off-target activity through the recognition of near-cognate DNA sequences to their target sites, resulting in collateral damage to the genome in the form of local mutagenesis or genomic rearrangements. For therapeutic genome-editing applications with these classes of programmable enzymes, it is essential to measure and limit genome-wide off-target activity. Herein, we discuss the key determinants of off-target activity for these systems. We describe various cell-based and cell-free methods for identifying genome-wide off-target sites and diverse strategies that have been developed for reducing the off-target activity of programmable gene-editing enzymes.
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7.
Production of protein-based polymers in Pichia pastoris.
Werten, MWT, Eggink, G, Cohen Stuart, MA, de Wolf, FA
Biotechnology advances. 2019;(5):642-666
Abstract
Materials science and genetic engineering have joined forces over the last three decades in the development of so-called protein-based polymers. These are proteins, typically with repetitive amino acid sequences, that have such physical properties that they can be used as functional materials. Well-known natural examples are collagen, silk, and elastin, but also artificial sequences have been devised. These proteins can be produced in a suitable host via recombinant DNA technology, and it is this inherent control over monomer sequence and molecular size that renders this class of polymers of particular interest to the fields of nanomaterials and biomedical research. Traditionally, Escherichia coli has been the main workhorse for the production of these polymers, but the methylotrophic yeast Pichia pastoris is finding increased use in view of the often high yields and potential bioprocessing benefits. We here provide an overview of protein-based polymers produced in P. pastoris. We summarize their physicochemical properties, briefly note possible applications, and detail their biosynthesis. Some challenges that may be faced when using P. pastoris for polymer production are identified: (i) low yields and poor process control in shake flask cultures; i.e., the need for bioreactors, (ii) proteolytic degradation, and (iii) self-assembly in vivo. Strategies to overcome these challenges are discussed, which we anticipate will be of interest also to readers involved in protein expression in P. pastoris in general.
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8.
Engineering of L-amino acid deaminases for the production of α-keto acids from L-amino acids.
Nshimiyimana, P, Liu, L, Du, G
Bioengineered. 2019;(1):43-51
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Abstract
α-keto acids are organic compounds that contain an acid group and a ketone group. L-amino acid deaminases are enzymes that catalyze the oxidative deamination of amino acids for the formation of their corresponding α-keto acids and ammonia. α-keto acids are synthesized industrially via chemical processes that are costly and use harsh chemicals. The use of the directed evolution technique, followed by the screening and selection of desirable variants, to evolve enzymes has proven to be an effective way to engineer enzymes with improved performance. This review presents recent studies in which the directed evolution technique was used to evolve enzymes, with an emphasis on L-amino acid deaminases for the whole-cell biocatalysts production of α-keto acids from their corresponding L-amino acids. We discuss and highlight recent cases where the engineered L-amino acid deaminases resulted in an improved production yield of phenylpyruvic acid, α-ketoisocaproate, α-ketoisovaleric acid, α-ketoglutaric acid, α-keto-γ-methylthiobutyric acid, and pyruvate.
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Engineering of the Dsb (disulfide bond) proteins - contribution towards understanding their mechanism of action and their applications in biotechnology and medicine.
Banaś, AM, Bocian-Ostrzycka, KM, Jagusztyn-Krynicka, EK
Critical reviews in microbiology. 2019;(4):433-450
Abstract
The Dsb protein family in prokaryotes catalyzes the generation of disulfide bonds between thiol groups of cysteine residues in nascent proteins, ensuring their proper three-dimensional structure; these bonds are crucial for protein stability and function. The first Dsb protein, Escherichia coli DsbA, was described in 1991. Since then, many details of the bond-formation process have been described through microbiological, biochemical, biophysical and bioinformatics strategies. Research with the model microorganism E. coli and many other bacterial species revealed an enormous diversity of bond-formation mechanisms. Research using Dsb protein engineering has significantly helped to reveal details of the disulfide bond formation. The first part of this review presents the research that led to understanding the mechanism of action of DsbA proteins, which directly transfer their own disulfide into target proteins. The second part concentrates on the mechanism of electron transport through the cell cytoplasmic membrane. Third and lastly, the review discusses the contribution of this research towards new antibacterial agents.
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10.
Engineering the orange carotenoid protein for applications in synthetic biology.
Dominguez-Martin, MA, Kerfeld, CA
Current opinion in structural biology. 2019;:110-117
Abstract
The Orange Carotenoid Protein (OCP) is a water-soluble protein that enables photoprotective energy quenching in cyanobacteria. Structurally, the OCP is modular, consisting of an all-helical N-terminal domain (NTD), and a mixed α-β C-terminal domain (CTD). A non-covalently associated carotenoid spans the two domains. Upon photoactivation, the carotenoid translocates into the NTD, causing a visible color change from orange to red, to activate the OCP as a quencher of excess captured energy. The recent identification of new families of the OCP with diverse activation and quenching properties, and the discovery of carotenoid-binding proteins that are homologs to either the discrete NTD or CTD, provide a tremendous repertoire of modules that can be used as building blocks for the design of custom photoswitchable proteins. Here based on recent foundational results, we describe potential ways that the OCP can be engineered to serve a wide range of applications in synthetic biology.