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1.
Enzyme Microb Technol ; 150: 109863, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489022

RESUMO

Protein-glutaminase (EC 3.5.1.44, PG) converts protein glutamine residues in proteins and peptides into glutamic acid residue, and markedly improves the solubility, emulsification, and foaming properties of food proteins. However, the source bacteria, Chryseobacterium proteolyticum, have low enzyme production ability, inefficient genetic operation, and high production cost. Therefore, it is critical to establish an efficient expression system for active PG. Here, combinatorial engineering was developed for high-yield production of PG in Bacillus subtilis. First, we evaluated different B. subtilis strains for PG self-activation. Then, combinatorial optimization involving promoters, signal peptides, and culture medium was applied to produce active recombinant PG in a B. subtilis expression system. Through combinatorial engineering, PG enzyme activity reached 3.23 U/mL in shaken-flask cultures. Active PG with the yield of 7.07 U/mL was obtained at 40 h by the PSecA-YdeJ combination in fed-batch fermentation, which is the highest yield of PG in existing reports.


Assuntos
Bacillus subtilis , Proteínas de Bactérias/biossíntese , Chryseobacterium , Glutaminase/biossíntese , Bacillus subtilis/metabolismo , Chryseobacterium/enzimologia , Fermentação , Engenharia de Proteínas , Sinais Direcionadores de Proteínas
2.
Int J Mol Sci ; 22(17)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34502016

RESUMO

Antisense peptide technology (APT) is based on a useful heuristic algorithm for rational peptide design. It was deduced from empirical observations that peptides consisting of complementary (sense and antisense) amino acids interact with higher probability and affinity than the randomly selected ones. This phenomenon is closely related to the structure of the standard genetic code table, and at the same time, is unrelated to the direction of its codon sequence translation. The concept of complementary peptide interaction is discussed, and its possible applications to diagnostic tests and bioengineering research are summarized. Problems and difficulties that may arise using APT are discussed, and possible solutions are proposed. The methodology was tested on the example of SARS-CoV-2. It is shown that the CABS-dock server accurately predicts the binding of antisense peptides to the SARS-CoV-2 receptor binding domain without requiring predefinition of the binding site. It is concluded that the benefits of APT outweigh the costs of random peptide screening and could lead to considerable savings in time and resources, especially if combined with other computational and immunochemical methods.


Assuntos
Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , Algoritmos , Sequência de Aminoácidos/genética , Sítios de Ligação/genética , COVID-19/sangue , COVID-19/virologia , Humanos , Imunoquímica/métodos , Simulação de Acoplamento Molecular , Peptídeos/genética , Ligação Proteica/genética , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/metabolismo
3.
Nat Commun ; 12(1): 4946, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400632

RESUMO

5-Hydroxymethylfurfural (HMF) has emerged as a crucial bio-based chemical building block in the drive towards developing materials from renewable resources, due to its direct preparation from sugars and its readily diversifiable scaffold. A key obstacle in transitioning to bio-based plastic production lies in meeting the necessary industrial production efficiency, particularly in the cost-effective conversion of HMF to valuable intermediates. Toward addressing the challenge of developing scalable technology for oxidizing crude HMF to more valuable chemicals, here we report coordinated reaction and enzyme engineering to provide a galactose oxidase (GOase) variant with remarkably high activity toward HMF, improved O2 binding and excellent productivity (>1,000,000 TTN). The biocatalyst and reaction conditions presented here for GOase catalysed selective oxidation of HMF to 2,5-diformylfuran offers a productive blueprint for further development, giving hope for the creation of a biocatalytic route to scalable production of furan-based chemical building blocks from sustainable feedstocks.


Assuntos
Furaldeído/análogos & derivados , Furaldeído/metabolismo , Galactose Oxidase/genética , Galactose Oxidase/metabolismo , Engenharia de Proteínas , Biocatálise , Catálise , Domínio Catalítico , Furanos , Galactose Oxidase/química , Mutagênese , Oxirredução
4.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360993

RESUMO

The ferroxidase ceruloplasmin (CP) plays a crucial role in iron homeostasis in vertebrates together with the iron exporter ferroportin. Mutations in the CP gene give rise to aceruloplasminemia, a rare neurodegenerative disease for which no cure is available. Many aspects of the (patho)physiology of CP are still unclear and would benefit from the availability of recombinant protein for structural and functional studies. Furthermore, recombinant CP could be evaluated for enzyme replacement therapy for the treatment of aceruloplasminemia. We report the production and preliminary characterization of high-quality recombinant human CP in glycoengineered Pichia pastoris SuperMan5. A modified yeast strain lacking the endogenous ferroxidase has been generated and employed as host for heterologous expression of the secreted isoform of human CP. Highly pure biologically active protein has been obtained by an improved two-step purification procedure. Glycan analysis indicates that predominant glycoforms HexNAc2Hex8 and HexNAc2Hex11 are found at Asn119, Asn378, and Asn743, three of the canonical four N-glycosylation sites of human CP. The availability of high-quality recombinant human CP represents a significant advancement in the field of CP biology. However, productivity needs to be increased and further careful glycoengineering of the SM5 strain is mandatory in order to evaluate the possible therapeutic use of the recombinant protein for enzyme replacement therapy of aceruloplasminemia patients.


Assuntos
Ceruloplasmina/genética , Microbiologia Industrial/métodos , Engenharia de Proteínas/métodos , Saccharomycetales/genética , Ceruloplasmina/metabolismo , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales/metabolismo
5.
Science ; 373(6553): 413-419, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34437114

RESUMO

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) regulates metabolism in response to the cellular energy states. Under energy stress, AMP stabilizes the active AMPK conformation, in which the kinase activation loop (AL) is protected from protein phosphatases, thus keeping the AL in its active, phosphorylated state. At low AMP:ATP (adenosine triphosphate) ratios, ATP inhibits AMPK by increasing AL dynamics and accessibility. We developed conformation-specific antibodies to trap ATP-bound AMPK in a fully inactive, dynamic state and determined its structure at 3.5-angstrom resolution using cryo-electron microscopy. A 180° rotation and 100-angstrom displacement of the kinase domain fully exposes the AL. On the basis of the structure and supporting biophysical data, we propose a multistep mechanism explaining how adenine nucleotides and pharmacological agonists modulate AMPK activity by altering AL phosphorylation and accessibility.


Assuntos
Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/imunologia , Proteínas Quinases Ativadas por AMP/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Humanos , Fragmentos Fab das Imunoglobulinas , Modelos Moleculares , Fosforilação , Conformação Proteica , Domínios Proteicos , Engenharia de Proteínas
6.
Viruses ; 13(8)2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34452461

RESUMO

The COVID-19 pandemic, caused by SARS-CoV-2, has rapidly spread to more than 222 countries and has put global public health at high risk. The world urgently needs cost-effective and safe SARS-CoV-2 vaccines, antiviral, and therapeutic drugs to control it. In this study, we engineered the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein and produced it in the plant Nicotiana benthamiana in a glycosylated and deglycosylated form. Expression levels of both glycosylated (gRBD) and deglycosylated (dRBD) RBD were greater than 45 mg/kg fresh weight. The purification yields were 22 mg of pure protein/kg of plant biomass for gRBD and 20 mg for dRBD, which would be sufficient for commercialization of these vaccine candidates. The purified plant-produced RBD protein was recognized by an S protein-specific monoclonal antibody, demonstrating specific reactivity of the antibody to the plant-produced RBD proteins. The SARS-CoV-2 RBD showed specific binding to angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor. In mice, the plant-produced RBD antigens elicited high titers of antibodies with a potent virus-neutralizing activity. To our knowledge, this is the first report demonstrating that mice immunized with plant-produced deglycosylated RBD form elicited high titer of RBD-specific antibodies with potent neutralizing activity against SARS-CoV-2 infection. Thus, obtained data support that plant-produced glycosylated and in vivo deglycosylated RBD antigens, developed in this study, are promising vaccine candidates for the prevention of COVID-19.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Glicosilação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Plantas Geneticamente Modificadas , Ligação Proteica , Domínios Proteicos , Engenharia de Proteínas , Estabilidade Proteica , Receptores de Coronavírus/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Tabaco/genética , Tabaco/metabolismo , Células Vero
7.
Methods Mol Biol ; 2352: 227-236, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34324190

RESUMO

Gene expression regulation by transcription factors plays a central role in determining and maintaining cell fate during normal development as well as induced cell fate reprogramming. Induction of cell identity-determining gene regulatory networks by reprogramming factors that act as transcriptional activators is key to induce desired cell fates. Conversely, repression of unwanted genetic programs by transcriptional repressors is equally important to ensure cell fate fidelity. Here we describe engineering techniques to create fusion proteins that allow exploration of the major transcriptional contribution (activation or repression) of specific neuronal reprogramming factors during direct cell fate conversion. This method can be extended to every reprogramming regime to enable the functional categorization of any transcription factor.


Assuntos
Diferenciação Celular , Técnicas de Reprogramação Celular , Reprogramação Celular , Engenharia de Proteínas , Fatores de Transcrição/genética , Diferenciação Celular/genética , Clonagem Molecular , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Fatores de Transcrição/metabolismo
8.
Nat Commun ; 12(1): 4219, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244505

RESUMO

Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , RNA Guia/genética , Staphylococcus/enzimologia , Animais , Proteína 9 Associada à CRISPR/isolamento & purificação , Linhagem Celular Tumoral , Modelos Animais de Doenças , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Camundongos , Parvovirinae/genética , Engenharia de Proteínas , Ribonucleases , Staphylococcus/genética , Especificidade por Substrato , Síndromes de Usher/genética , Síndromes de Usher/terapia
9.
Methods Mol Biol ; 2312: 15-33, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228282

RESUMO

Synthetic receptors control cell behavior in response to environmental stimuli for applications in basic research and cell therapy. However, the integration of synthetic receptors in unexplored contexts is cumbersome, especially for nonspecialist laboratories. Here, I provide a detailed protocol on how to use receptors of the generalized extracellular molecule sensor (GEMS) platform. GEMS is a modular receptor system that can be adapted to sense molecules of choice by using affinity domains that dimerize in response to the target. GEMS consist of an erythropoietin receptor scaffold that has been mutated to no longer bind to erythropoietin. N-terminal fusions with affinity domains, such as single chain variable fragments (scFvs), that bind to two epitopes on the same target activate the receptor. The intracellular receptor domain can be chosen from several signal transduction domains of single-pass transmembrane receptors to activate endogenous signaling pathways. As of now, GEMS have been used for sensing prostate specific antigen (PSA), the synthetic azo dye RR120, caffeine, nicotine, rapamycin, the SunTag peptide, and a de novo designed protein displaying two viral epitopes. The tested intracellular domains were derived from FGFR1, IL-6RB, and VEGFR2, and were used to drive transgene expression from reporter plasmids responsive to the endogenous transcription factors STAT3, NFAT, NF-κB, and a synthetic transcription factor activated by the MAPK pathway. In this protocol, I focus on transient transfections of HEK293T cells and include several general notes about cell handling. While the described methods are optimized for experiments with GEMS, most of the described techniques are general procedures to set up synthetic biology experiments in mammalian cell culture. I outline how to generate stable cell lines and share tips on how to adapt GEMS for new ligands. The main objective of this protocol is to make the GEMS technology accessible also to nonspecialist laboratories to facilitate the use of synthetic receptors in new research contexts.


Assuntos
Técnicas Biossensoriais , Engenharia Celular , Engenharia de Proteínas , Receptores Artificiais/genética , Receptores da Eritropoetina/genética , Anticorpos de Cadeia Única/genética , Biologia Sintética , Afinidade de Anticorpos , Receptor gp130 de Citocina/genética , Epitopos , Genes Reporter , Células HEK293 , Humanos , Ligantes , Mutação , Domínios e Motivos de Interação entre Proteínas , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptores Artificiais/metabolismo , Receptores da Eritropoetina/metabolismo , Transdução de Sinais , Anticorpos de Cadeia Única/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
10.
Methods Mol Biol ; 2312: 277-285, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228296

RESUMO

There are increasing evidence and growing interest in the relationship between protein aggregates/phase separation and various human diseases, especially neurodegenerative diseases. However, we do not entirely comprehend how aggregates generate or the clearance network of chaperones, proteasomes, ubiquitin ligases, and other factors interact with aggregates. Here, we describe chemically controllable systems compose with a genetically engineered cell and a small drug that enables us to rapidly induce protein aggregates' formation by withdrawing the small molecule. This trigger does not activate global stress responses induced by stimuli, such as proteasome inhibitors or heat shock. This method can produce aggregates in a specific compartment and diverse experimental systems, including live animals.


Assuntos
Engenharia Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Engenharia de Proteínas , Proteínas de Ligação a Tacrolimo/genética , Animais , Técnicas de Cultura de Células , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Camundongos , Microscopia de Fluorescência , Mutação , Células NIH 3T3 , Agregados Proteicos , Estabilidade Proteica , Proteostase , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Transfecção
11.
Methods Mol Biol ; 2312: 287-300, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228297

RESUMO

Artificial metalloenzymes, constructed by incorporating a synthetic catalyst into the internal spaces of a protein scaffold, can perform noncanonical chemical transformations that are not possible using natural enzymes. The addition of cell-permeable modules to artificial metalloenzymes allows for noncanonical catalysis to be implemented as a function of mammalian cells. In this chapter, we describe a protocol for controlling cellular function through a cascade consisting of an artificial metalloenzyme and a gene-circuit engineered via synthetic biology.


Assuntos
Engenharia Celular , Enzimas/metabolismo , Metaloproteínas/metabolismo , Engenharia de Proteínas , Biologia Sintética , Biotina/química , Catálise , Técnicas de Cultura de Células , Enzimas/genética , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Humanos , Metaloproteínas/genética , Estreptavidina/química , Especificidade por Substrato , Transfecção
12.
Methods Mol Biol ; 2315: 43-57, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302669

RESUMO

Protein engineering can yield new molecular tools for nanotechnology and therapeutic applications through modulating physiochemical and biological properties. Engineering membrane proteins is especially attractive because they perform key cellular processes including transport, nutrient uptake, removal of toxins, respiration, motility, and signaling. In this chapter, we describe two protocols for membrane protein engineering with the Rosetta software: (1) ΔΔG calculations for single point mutations and (2) sequence optimization in different membrane lipid compositions. These modular protocols are easily adaptable for more complex problems and serve as a foundation for efficient membrane protein engineering calculations.


Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Membranas/química , Membranas/metabolismo , Engenharia de Proteínas/métodos , Transporte Biológico/fisiologia , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Modelos Moleculares , Software
13.
Methods Mol Biol ; 2315: 59-70, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302670

RESUMO

The rational in silico design of interface mutations within protein complexes is a synthetic biology tool that enables-when introduced into biological systems-the artificial rewiring of biological pathways. Here we describe the three-dimensional structure-based design of "rewiring" mutations using the FoldX force field. Specifically, we provide the protocol for the design and selection of interface mutations in three Ras-effector complex structures (PDB entries 3KUD, 4K81, and 6AMB). Ras mutations that impair binding to some but not all interacting partners are selected.


Assuntos
Transdução de Sinais/genética , Simulação por Computador , Modelos Moleculares , Mutação/genética , Ligação Proteica/genética , Engenharia de Proteínas/métodos , Proteínas ras/genética
14.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298982

RESUMO

Engineering of biomimetic motives have emerged as promising approaches to improving cells' binding properties of biomaterials for tissue engineering and regenerative medicine. In this study, a bio-adhesive ligand including cell-binding domains of human fibronectin (FN) was engineered using recombinant protein technology, a major extracellular matrix (ECM) protein that interacts with a variety of integrins cell-surface's receptors and other ECM proteins through specific binding domains. 9th and 10th fibronectin type III repeat containing Arginine-Glycine-Aspartic acid (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) synergic site (FNIII9-10) were expressed in fusion with a Colored Multi Affinity Tag (CMAT) to develop a simplified production and characterization process. A recombinant fragment was produced in the bacterial system using E. coli with high yield purified protein by double affinity chromatography. Bio-adhesive surfaces were developed by passive coating of produced fragment onto non adhesive surfaces model. The recombinant fusion protein (CMAT-FNIII9/10) demonstrated an accurate monitoring capability during expression purification and adsorption assay. Finally, biological activity of recombinant FNIII9/10 was validated by cellular adhesion assay. Binding to α5ß1 integrins were successfully validated using a produced fragment as a ligand. These results are robust supports to the rational development of bioactivation strategies for biomedical and biotechnological applications.


Assuntos
Marcadores de Afinidade , Materiais Biomiméticos , Fibronectinas , Oligopeptídeos , Fragmentos de Peptídeos , Proteínas Recombinantes de Fusão/química , Adsorção , Adesão Celular , Materiais Revestidos Biocompatíveis , Escherichia coli , Matriz Extracelular/química , Vetores Genéticos , Integrina alfa5beta1/metabolismo , Ligantes , Espectrometria de Massas , Poliestirenos , Ligação Proteica , Domínios Proteicos , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo
15.
Sheng Wu Gong Cheng Xue Bao ; 37(6): 1845-1857, 2021 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-34227280

RESUMO

Non-ribosomal peptide synthetases catalyze the biosynthesis of structurally and functionally diverse non-ribosomal peptide natural products, which have broad applications in pharmaceutical, agricultural, and industrial sectors. Engineered non-ribosomal peptide synthetases can be used to produce novel non-ribosomal peptides through combinatorial biosynthesis. This conforms to the concept of green chemistry, thus attracts increasing attention across the world. Herein, three different engineering strategies were summarized, and recent advances in this field were reviewed.


Assuntos
Produtos Biológicos , Peptídeo Sintases , Engenharia de Proteínas , Peptídeo Sintases/genética , Peptídeos
16.
Sheng Wu Gong Cheng Xue Bao ; 37(6): 1919-1930, 2021 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-34227285

RESUMO

Glycosidases are widely used in food and pharmaceutical industries due to its ability to hydrolyze the glycosidic bonds of various sugar-containing compounds including glycosides, oligosaccharides and polysaccharides to generate derivatives with important physiological and pharmacological activity. While glycosidases often need to be used under high temperature to improve reaction efficiency and reduce contamination, most glycosidases are mesophilic enzymes with low activity under industrial production conditions. It is therefore critical to improve the thermo-stability of glycosidases. This review summarizes the recent advances achieved in engineering the thermo-stability of glycosidases using strategies such as directed evolution, rational design and semi-rational design. We also compared the pros and cons of various techniques and discussed the future prospects in this area.


Assuntos
Glicosídeo Hidrolases , Engenharia de Proteínas , Glicosídeo Hidrolases/genética , Oligossacarídeos , Polissacarídeos
17.
Sheng Wu Gong Cheng Xue Bao ; 37(6): 2105-2115, 2021 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-34227297

RESUMO

Triterpenoids are a class of natural products of great commercial value that are widely used in pharmaceutical, health care and cosmetic industries. The biosynthesis of triterpenoids relies on the efficient synthesis of squalene epoxide, which is synthesized from the NADPH dependent oxidation of squalene catalyzed by squalene epoxidase. We screened squalene epoxidases derived from different species, and found the truncated squalene epoxidase from Rattus norvegicus (RnSETC) showed the highest activity in engineered Escherichia coli. Further examination of the effect of endogenous cytochrome P450 reductase like (CPRL) proteins showed that overexpression of NADH: quinone oxidoreductase (WrbA) under Lac promoter in a medium-copy number plasmid increased the production of squalene epoxide by nearly 2.5 folds. These results demonstrated that the constructed pathway led to the production of squalene epoxide, an important precursor for the biosynthesis of triterpenoids.


Assuntos
Esqualeno Mono-Oxigenase , Esqualeno , Animais , Escherichia coli/genética , NADPH-Ferri-Hemoproteína Redutase , Engenharia de Proteínas , Ratos , Proteínas Repressoras , Esqualeno Mono-Oxigenase/genética
18.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206616

RESUMO

The biophysical properties of therapeutic antibodies influence their manufacturability, efficacy, and safety. To develop an anti-cancer antibody, we previously generated a human monoclonal antibody (Ab417) that specifically binds to L1 cell adhesion molecule with a high affinity, and we validated its anti-tumor activity and mechanism of action in human cholangiocarcinoma xenograft models. In the present study, we aimed to improve the biophysical properties of Ab417. We designed 20 variants of Ab417 with reduced aggregation propensity, less potential post-translational modification (PTM) motifs, and the lowest predicted immunogenicity using computational methods. Next, we constructed these variants to analyze their expression levels and antigen-binding activities. One variant (Ab612)-which contains six substitutions for reduced surface hydrophobicity, removal of PTM, and change to the germline residue-exhibited an increased expression level and antigen-binding activity compared to Ab417. In further studies, compared to Ab417, Ab612 showed improved biophysical properties, including reduced aggregation propensity, increased stability, higher purification yield, lower pI, higher affinity, and greater in vivo anti-tumor efficacy. Additionally, we generated a highly productive and stable research cell bank (RCB) and scaled up the production process to 50 L, yielding 6.6 g/L of Ab612. The RCB will be used for preclinical development of Ab612.


Assuntos
Anticorpos Monoclonais/química , Modelos Moleculares , Molécula L1 de Adesão de Célula Nervosa/química , Engenharia de Proteínas , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Afinidade de Anticorpos , Células CHO , Fenômenos Químicos , Cricetulus , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Engenharia de Proteínas/métodos , Estabilidade Proteica , Termodinâmica
19.
Methods Enzymol ; 656: 375-428, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34325793

RESUMO

Over the past decade, harnessing the cellular protein synthesis machinery to incorporate non-canonical amino acids (ncAAs) into tailor-made peptides has significantly advanced many aspects of molecular science. More recently, groundbreaking progress in our ability to engineer this machinery for improved ncAA incorporation has led to significant enhancements of this powerful tool for biology and chemistry. By revealing the molecular basis for the poor or improved incorporation of ncAAs, mechanistic studies of ncAA incorporation by the protein synthesis machinery have tremendous potential for informing and directing such engineering efforts. In this chapter, we describe a set of complementary biochemical and single-molecule fluorescence assays that we have adapted for mechanistic studies of ncAA incorporation. Collectively, these assays provide data that can guide engineering of the protein synthesis machinery to expand the range of ncAAs that can be incorporated into peptides and increase the efficiency with which they can be incorporated, thereby enabling the full potential of ncAA mutagenesis technology to be realized.


Assuntos
Aminoácidos , Aminoacil-tRNA Sintetases , Aminoácidos/genética , Aminoacil-tRNA Sintetases/genética , Mutagênese , Biossíntese de Proteínas , Engenharia de Proteínas
20.
J Chem Inf Model ; 61(7): 3463-3476, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34260225

RESUMO

Data-driven strategies are gaining increased attention in protein engineering due to recent advances in access to large experimental databanks of proteins, next-generation sequencing (NGS), high-throughput screening (HTS) methods, and the development of artificial intelligence algorithms. However, the reliable prediction of beneficial amino acid substitutions, their combination, and the effect on functional properties remain the most significant challenges in protein engineering, which is applied to develop proteins and enzymes for biocatalysis, biomedicine, and life sciences. Here, we present a general-purpose framework (PyPEF: pythonic protein engineering framework) for performing data-driven protein engineering using machine learning methods combined with techniques from signal processing and statistical physics. PyPEF guides the identification and selection of beneficial proteins of a defined sequence space by systematically or randomly exploring the fitness of variants and by sampling random evolution pathways. The performance of PyPEF was evaluated concerning its predictive accuracy and throughput on four public protein and enzyme data sets using common regression models. It was proved that the program could efficiently predict the fitness of protein sequences for different target properties (predictive models with coefficient of determination values ranging from 0.58 to 0.92). By combining machine learning and protein evolution, PyPEF enabled the screening of proteins with various functions, reaching a screening capacity of more than 500,000 protein sequence variants in the timeframe of only a few minutes on a personal computer. PyPEF displayed significant accuracies on four public data sets (different proteins and properties) and underlined the potential of integrating data-driven technologies for covering different philosophies by either predicting the fitness of the variants to the highest accuracy accounting for epistatic effects or capturing the general trend of introduced mutations on the fitness in directed protein evolution campaigns. In essence, PyPEF can provide a powerful solution to current sequence exploration and combinatorial problems faced in protein engineering through exhaustive in silico screening of the sequence space.


Assuntos
Inteligência Artificial , Evolução Molecular Direcionada , Sequência de Aminoácidos , Aprendizado de Máquina , Engenharia de Proteínas
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