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Science (New York, N.Y.) Oct 2022Although deep learning has revolutionized protein structure prediction, almost all experimentally characterized de novo protein designs have been generated using...
Although deep learning has revolutionized protein structure prediction, almost all experimentally characterized de novo protein designs have been generated using physically based approaches such as Rosetta. Here, we describe a deep learning-based protein sequence design method, ProteinMPNN, that has outstanding performance in both in silico and experimental tests. On native protein backbones, ProteinMPNN has a sequence recovery of 52.4% compared with 32.9% for Rosetta. The amino acid sequence at different positions can be coupled between single or multiple chains, enabling application to a wide range of current protein design challenges. We demonstrate the broad utility and high accuracy of ProteinMPNN using x-ray crystallography, cryo-electron microscopy, and functional studies by rescuing previously failed designs, which were made using Rosetta or AlphaFold, of protein monomers, cyclic homo-oligomers, tetrahedral nanoparticles, and target-binding proteins.
Topics: Amino Acid Sequence; Cryoelectron Microscopy; Crystallography, X-Ray; Deep Learning; Protein Conformation; Protein Engineering; Proteins
PubMed: 36108050
DOI: 10.1126/science.add2187 -
Methods in Molecular Biology (Clifton,... 2015The method of displaying recombinant proteins on the surface of Saccharomyces cerevisiae via genetic fusion to an abundant cell wall protein, a technology known as yeast... (Review)
Review
The method of displaying recombinant proteins on the surface of Saccharomyces cerevisiae via genetic fusion to an abundant cell wall protein, a technology known as yeast surface display, or simply, yeast display, has become a valuable protein engineering tool for a broad spectrum of biotechnology and biomedical applications. This review focuses on the use of yeast display for engineering protein affinity, stability, and enzymatic activity. Strategies and examples for each protein engineering goal are discussed. Additional applications of yeast display are also briefly presented, including protein epitope mapping, identification of protein-protein interactions, and uses of displayed proteins in industry and medicine.
Topics: Cell Surface Display Techniques; Epitope Mapping; Protein Binding; Protein Engineering; Protein Stability; Recombinant Proteins; Saccharomyces cerevisiae
PubMed: 26060074
DOI: 10.1007/978-1-4939-2748-7_8 -
Nature Methods Dec 2019Rational protein engineering requires a holistic understanding of protein function. Here, we apply deep learning to unlabeled amino-acid sequences to distill the...
Rational protein engineering requires a holistic understanding of protein function. Here, we apply deep learning to unlabeled amino-acid sequences to distill the fundamental features of a protein into a statistical representation that is semantically rich and structurally, evolutionarily and biophysically grounded. We show that the simplest models built on top of this unified representation (UniRep) are broadly applicable and generalize to unseen regions of sequence space. Our data-driven approach predicts the stability of natural and de novo designed proteins, and the quantitative function of molecularly diverse mutants, competitively with the state-of-the-art methods. UniRep further enables two orders of magnitude efficiency improvement in a protein engineering task. UniRep is a versatile summary of fundamental protein features that can be applied across protein engineering informatics.
Topics: Amino Acid Sequence; Deep Learning; Mutation; Protein Engineering; Protein Stability
PubMed: 31636460
DOI: 10.1038/s41592-019-0598-1 -
Biochimie Oct 2020An antibody's stability greatly influences its performance (i.e. its specificity and affinity). Thus, stability is a major issue for researchers and manufacturers,... (Review)
Review
An antibody's stability greatly influences its performance (i.e. its specificity and affinity). Thus, stability is a major issue for researchers and manufacturers, especially with the increasing use of antibodies in therapeutics, diagnostics and rapid analytical platforms. Here we review antibody stability under five headings: (i) measurement techniques; (ii) stability issues in expression and production (expression, proteolysis, aggregation); (iii) effects of antibody format and engineering on stability and (iv) formulation, drying and storage conditions. We consider more than 100 sources, including patents, and conclude with (v) recommendations to promote antibody stability.
Topics: Animals; Antibodies; Cold Temperature; Drug Compounding; Drug Storage; Humans; Protein Engineering; Protein Stability
PubMed: 32891698
DOI: 10.1016/j.biochi.2020.08.019 -
Current Opinion in Structural Biology Aug 2022
Topics: Protein Engineering
PubMed: 35872560
DOI: 10.1016/j.sbi.2022.102437 -
MAbs 2020Monoclonal antibodies play an increasingly important role for the development of new drugs across multiple therapy areas. The term 'developability' encompasses the...
Monoclonal antibodies play an increasingly important role for the development of new drugs across multiple therapy areas. The term 'developability' encompasses the feasibility of molecules to successfully progress from discovery to development via evaluation of their physicochemical properties. These properties include the tendency for self-interaction and aggregation, thermal stability, colloidal stability, and optimization of their properties through sequence engineering. Selection of the best antibody molecule based on biological function, efficacy, safety, and developability allows for a streamlined and successful CMC phase. An efficient and practical high-throughput developability workflow (100 s-1,000 s of molecules) implemented during early antibody generation and screening is crucial to select the best lead candidates. This involves careful assessment of critical developability parameters, combined with binding affinity and biological properties evaluation using small amounts of purified material (<1 mg), as well as an efficient data management and database system. Herein, a panel of 152 various human or humanized monoclonal antibodies was analyzed in biophysical property assays. Correlations between assays for different sets of properties were established. We demonstrated in two case studies that physicochemical properties and key assay endpoints correlate with key downstream process parameters. The workflow allows the elimination of antibodies with suboptimal properties and a rank ordering of molecules for further evaluation early in the candidate selection process. This enables any further engineering for problematic sequence attributes without affecting program timelines.
Topics: Antibodies, Monoclonal; Drug Discovery; Humans; Protein Engineering; Workflow
PubMed: 32249670
DOI: 10.1080/19420862.2020.1743053 -
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 -
Science (New York, N.Y.) Nov 2019Adeno-associated virus (AAV) capsids can deliver transformative gene therapies, but our understanding of AAV biology remains incomplete. We generated the complete...
Adeno-associated virus (AAV) capsids can deliver transformative gene therapies, but our understanding of AAV biology remains incomplete. We generated the complete first-order AAV2 capsid fitness landscape, characterizing all single-codon substitutions, insertions, and deletions across multiple functions relevant for in vivo delivery. We discovered a frameshifted gene in the VP1 region that expresses a membrane-associated accessory protein that limits AAV production through competitive exclusion. Mutant biodistribution revealed the importance of both surface-exposed and buried residues, with a few phenotypic profiles characterizing most variants. Finally, we algorithmically designed and experimentally verified a diverse in vivo targeted capsid library with viability far exceeding random mutagenesis approaches. These results demonstrate the power of systematic mutagenesis for deciphering complex genomes and the potential of empirical machine-guided protein engineering.
Topics: Capsid; Capsid Proteins; Dependovirus; Genes, Viral; Genetic Therapy; HEK293 Cells; Humans; Mutagenesis, Insertional; Protein Engineering; Sequence Deletion; Transfection
PubMed: 31780559
DOI: 10.1126/science.aaw2900 -
Advances in Experimental Medicine and... 2019Our 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... (Review)
Review
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.
Topics: Allosteric Regulation; Mutagenesis, Site-Directed; Protein Engineering; Proteins
PubMed: 31707711
DOI: 10.1007/978-981-13-8719-7_15 -
Protein Engineering, Design & Selection... Jul 2016A popular and successful strategy in semi-rational design of protein stability is the use of evolutionary information encapsulated in homologous protein sequences.... (Review)
Review
A popular and successful strategy in semi-rational design of protein stability is the use of evolutionary information encapsulated in homologous protein sequences. Consensus design is based on the hypothesis that at a given position, the respective consensus amino acid contributes more than average to the stability of the protein than non-conserved amino acids. Here, we review the consensus design approach, its theoretical underpinnings, successes, limitations and challenges, as well as providing a detailed guide to its application in protein engineering.
Topics: Amino Acid Sequence; Animals; Humans; Informatics; Protein Engineering; Protein Stability; Proteins; Sequence Alignment; Temperature; Thermodynamics
PubMed: 27274091
DOI: 10.1093/protein/gzw015