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Pharmacological Reviews Apr 2020Ubiquitin (UB) transfer cascades consisting of E1, E2, and E3 enzymes constitute a complex network that regulates a myriad of biologic processes by modifying protein... (Review)
Review
Ubiquitin (UB) transfer cascades consisting of E1, E2, and E3 enzymes constitute a complex network that regulates a myriad of biologic processes by modifying protein substrates. Deubiquitinating enzymes (DUBs) reverse UB modifications or trim UB chains of diverse linkages. Additionally, many cellular proteins carry UB-binding domains (UBDs) that translate the signals encoded in UB chains to target proteins for degradation by proteasomes or in autophagosomes, as well as affect nonproteolytic outcomes such as kinase activation, DNA repair, and transcriptional regulation. Dysregulation of the UB transfer pathways and malfunctions of DUBs and UBDs play causative roles in the development of many diseases. A greater understanding of the mechanism of UB chain assembly and the signals encoded in UB chains should aid in our understanding of disease pathogenesis and guide the development of novel therapeutics. The recent flourish of protein-engineering approaches such as unnatural amino acid incorporation, protein semisynthesis by expressed protein ligation, and high throughput selection by phage and yeast cell surface display has generated designer proteins as powerful tools to interrogate cell signaling mediated by protein ubiquitination. In this study, we highlight recent achievements of protein engineering on mapping, probing, and manipulating UB transfer in the cell. SIGNIFICANCE STATEMENT: The post-translational modification of proteins with ubiquitin alters the fate and function of proteins in diverse ways. Protein engineering is fundamentally transforming research in this area, providing new mechanistic insights and allowing for the exploration of concepts that can potentially be applied to therapeutic intervention.
Topics: Animals; Deubiquitinating Enzymes; Humans; Protein Engineering; Ubiquitination; Ubiquitins
PubMed: 32107274
DOI: 10.1124/pr.118.015651 -
Current Opinion in Biotechnology Dec 2022Computational protein engineering has enabled the rational design of customized proteins, which has propelled both sequence-based and structure-based immunogen... (Review)
Review
Computational protein engineering has enabled the rational design of customized proteins, which has propelled both sequence-based and structure-based immunogen engineering and delivery. By discerning antigenic determinants of viral pathogens, computational methods have been implemented to successfully engineer representative viral strains able to elicit broadly neutralizing responses or present antigenic sites of viruses for focused immune responses. Combined with improvements in customizable nanoparticle design, immunogens are multivalently displayed to enhance immune responses. These rationally designed immunogens offer unique and powerful approaches to engineer vaccines for pathogens, which have eluded traditional approaches.
Topics: Antibodies, Neutralizing; Vaccines; Protein Engineering; AIDS Vaccines
PubMed: 36279815
DOI: 10.1016/j.copbio.2022.102821 -
FEBS Letters Jan 2014Improving the stability of proteins is an important goal in many biomedical and industrial applications. A logical approach is to emulate stabilizing molecular... (Review)
Review
Improving the stability of proteins is an important goal in many biomedical and industrial applications. A logical approach is to emulate stabilizing molecular interactions found in nature. Disulfide bonds are covalent interactions that provide substantial stability to many proteins and conform to well-defined geometric conformations, thus making them appealing candidates in protein engineering efforts. Disulfide engineering is the directed design of novel disulfide bonds into target proteins. This important biotechnological tool has achieved considerable success in a wide range of applications, yet the rules that govern the stabilizing effects of disulfide bonds are not fully characterized. Contrary to expectations, many designed disulfide bonds have resulted in decreased stability of the modified protein. We review progress in disulfide engineering, with an emphasis on the issue of stability and computational methods that facilitate engineering efforts.
Topics: Computers; Disulfides; Kinetics; Protein Engineering; Protein Stability; Proteins
PubMed: 24291258
DOI: 10.1016/j.febslet.2013.11.024 -
ACS Sensors Oct 2020Biological signaling pathways are underpinned by protein switches that sense and respond to molecular inputs. Inspired by nature, engineered protein switches have been... (Review)
Review
Biological signaling pathways are underpinned by protein switches that sense and respond to molecular inputs. Inspired by nature, engineered protein switches have been designed to directly transduce analyte binding into a quantitative signal in a simple, wash-free, homogeneous assay format. As such, they offer great potential to underpin point-of-need diagnostics that are needed across broad sectors to improve access, costs, and speed compared to laboratory assays. Despite this, protein switch assays are not yet in routine diagnostic use, and a number of barriers to uptake must be overcome to realize this potential. Here, we review the opportunities and challenges in engineering protein switches for rapid diagnostic tests. We evaluate how their design, comprising a recognition element, reporter, and switching mechanism, relates to performance and identify areas for improvement to guide further optimization. Recent modular switches that enable new analytes to be targeted without redesign are crucial to ensure robust and efficient development processes. The importance of translational steps toward practical implementation, including integration into a user-friendly device and thorough assay validation, is also discussed.
Topics: Biosensing Techniques; Diagnostic Tests, Routine; Protein Engineering; Proteins
PubMed: 33052043
DOI: 10.1021/acssensors.0c01831 -
Nucleic Acids Research Aug 2004We have developed a new primer design method based on the QuickChange site-directed mutagenesis protocol, which significantly improves the PCR amplification efficiency....
We have developed a new primer design method based on the QuickChange site-directed mutagenesis protocol, which significantly improves the PCR amplification efficiency. This design method minimizes primer dimerization and ensures the priority of primer-template annealing over primer self-pairing during the PCR. Several different multiple mutations (up to 7 bases) were successfully performed with this partial overlapping primer design in a variety of vectors ranging from 4 to 12 kb in length. In comparison, all attempts failed when using complete-overlapping primer pairs as recommended in the standard QuickChange protocol. Our protocol was further extended to site-saturation mutagenesis by introducing randomized codons. Our data indicated no specific sequence selection during library construction, with the randomized positions resulting in average occurrence of each base in each position. This method should be useful to facilitate the preparation of high-quality site saturation libraries.
Topics: DNA Primers; Directed Molecular Evolution; Electrophoresis, Agar Gel; Gene Library; Mutagenesis, Site-Directed; Polymerase Chain Reaction; Protein Engineering
PubMed: 15304544
DOI: 10.1093/nar/gnh110 -
Trends in Biotechnology Jan 2022The drastically increasing amount of plastic waste is causing an environmental crisis that requires innovative technologies for recycling post-consumer plastics to... (Review)
Review
The drastically increasing amount of plastic waste is causing an environmental crisis that requires innovative technologies for recycling post-consumer plastics to achieve waste valorization while meeting environmental quality goals. Biocatalytic depolymerization mediated by enzymes has emerged as an efficient and sustainable alternative for plastic treatment and recycling. A variety of plastic-degrading enzymes have been discovered from microbial sources. Meanwhile, protein engineering has been exploited to modify and optimize plastic-degrading enzymes. This review highlights the recent trends and up-to-date advances in mining novel plastic-degrading enzymes through state-of-the-art omics-based techniques and improving the enzyme catalytic efficiency and stability via various protein engineering strategies. Future research prospects and challenges are also discussed.
Topics: Biocatalysis; Plastics; Protein Engineering; Recycling
PubMed: 33676748
DOI: 10.1016/j.tibtech.2021.02.008 -
Current Opinion in Structural Biology Aug 2017The advent of next-generation sequencing (NGS) has revolutionized protein science, and the development of complementary methods enabling NGS-driven protein engineering... (Review)
Review
The advent of next-generation sequencing (NGS) has revolutionized protein science, and the development of complementary methods enabling NGS-driven protein engineering have followed. In general, these experiments address the functional consequences of thousands of protein variants in a massively parallel manner using genotype-phenotype linked high-throughput functional screens followed by DNA counting via deep sequencing. We highlight the use of information rich datasets to engineer protein molecular recognition. Examples include the creation of multiple dual-affinity Fabs targeting structurally dissimilar epitopes and engineering of a broad germline-targeted anti-HIV-1 immunogen. Additionally, we highlight the generation of enzyme fitness landscapes for conducting fundamental studies of protein behavior and evolution. We conclude with discussion of technological advances.
Topics: Animals; Epitope Mapping; High-Throughput Nucleotide Sequencing; Humans; Protein Engineering; Proteins
PubMed: 27886568
DOI: 10.1016/j.sbi.2016.11.001 -
Medecine Sciences : M/S Dec 2019Cytokines and biological toxins represent two potent classes of biomolecules that have long been explored for their potential as therapeutics. Considerable side effects... (Review)
Review
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.
Topics: Animals; Antibodies; Cytokines; Drug Evaluation, Preclinical; Humans; Immunotoxins; Protein Engineering; Recombinant Fusion Proteins
PubMed: 31903917
DOI: 10.1051/medsci/2019205 -
Bioengineered May 2017The evolution of natural modular proteins and domain swapping by protein engineers have shown the disruptive potential of non-homologous recombination to create proteins... (Review)
Review
The evolution of natural modular proteins and domain swapping by protein engineers have shown the disruptive potential of non-homologous recombination to create proteins with novel functions or traits. Bacteriophage endolysins, cellulosomes and polyketide synthases are 3 examples of natural modular proteins with each module having a dedicated function. These modular architectures have been created by extensive duplication, shuffling of domains and insertion/deletion of new domains. Protein engineers mimic these natural processes in vitro to create chimeras with altered properties or novel functions by swapping modules between different parental genes. Most domain swapping efforts are realized with traditional restriction and ligation techniques, which become particularly restrictive when either a large number of variants, or variants of proteins with multiple domains have to be constructed. Recent advances in homology-independent shuffling techniques increasingly address this need, but to realize the full potential of the synthetic biology of modular proteins a complete homology-independent method for both rational and random shuffling of modules from an unlimited number of parental genes is still needed.
Topics: Biomimetic Materials; Biosynthetic Pathways; Enzyme Activation; Enzymes; Protein Engineering; Substrate Specificity; Synthetic Biology
PubMed: 27645260
DOI: 10.1080/21655979.2016.1222993 -
Bioscience Reports Aug 2022In enzyme engineering, the main targets for enhancing properties are enzyme activity, stereoselective specificity, stability, substrate range, and the development of... (Review)
Review
In enzyme engineering, the main targets for enhancing properties are enzyme activity, stereoselective specificity, stability, substrate range, and the development of unique functions. With the advent of genetic code extension technology, non-natural amino acids (nnAAs) are able to be incorporated into proteins in a site-specific or residue-specific manner, which breaks the limit of 20 natural amino acids for protein engineering. Benefitting from this approach, numerous enzymes have been engineered with nnAAs for improved properties or extended functionality. In the present review, we focus on applications and strategies for using nnAAs in enzyme engineering. Notably, approaches to computational modelling of enzymes with nnAAs are also addressed. Finally, we discuss the bottlenecks that currently need to be addressed in order to realise the broader prospects of this genetic code extension technique.
Topics: Amino Acids; Cloning, Molecular; Protein Engineering; Proteins
PubMed: 35856922
DOI: 10.1042/BSR20220168