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1.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33468658

RESUMO

Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.


Assuntos
Diazometano/metabolismo , Histonas/genética , Lisina/metabolismo , Fases de Leitura Aberta , Peptídeos/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Bovinos , Cromatina/química , Cromatina/metabolismo , Diazometano/análogos & derivados , Regulação da Expressão Gênica , Loci Gênicos , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Células K562 , Lisina/análogos & derivados , Camundongos , Pan troglodytes , Peptídeos/metabolismo , Ligação Proteica/efeitos da radiação , Mapeamento de Interação de Proteínas , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Gênica/efeitos da radiação , Transgenes , Raios Ultravioleta
2.
Bioconjug Chem ; 30(1): 83-89, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30543420

RESUMO

Peptide hormone relaxin-2, a member of the insulin family of peptides, plays a key role in hemodynamics and renal function and has shown preclinical efficacy in multiple disease models, including acute heart failure, fibrosis, preeclampsia, and corneal wound healing. Recently, serelaxin, a recombinant version of relaxin-2, has been studied in a large phase 3 clinical trial (RELAX-AHF-2) for acute decompensated heart failure patients with disappointing outcome. The poor in vivo half-life of relaxin-2 may have limited its therapeutic efficacy and long-term cardiovascular benefit. Herein, we have developed a semisynthetic methodology and generated potent, fatty acid-conjugated relaxin analogs with long-acting pharmacokinetic (PK) profile in rodents. The enhanced PK properties translated into improved and long-lasting pharmacodynamic effect in pubic ligament elongation (PLE) studies. The resultant novel relaxin analog, R9-13, represents the first long-acting relaxin-2 analog and could potentially improve the clinical efficacy and outcome for this important peptide hormone. This semisynthetic methodology could also be applied to other cysteine-rich peptides and proteins for half-life extension.


Assuntos
Desenho de Fármacos , Lipídeos/química , Relaxina/química , Relaxina/uso terapêutico , Sequência de Aminoácidos , Animais , Meia-Vida , Insuficiência Cardíaca/tratamento farmacológico , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapêutico , Relaxina/farmacocinética
3.
Nature ; 562(7728): 600-604, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30323285

RESUMO

Mechanisms that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3-5 and transcription6. An important 'sensor' protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases.


Assuntos
Glicólise , Proteína 1 Associada a ECH Semelhante a Kelch/química , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Animais , Elementos de Resposta Antioxidante/genética , Arginina/química , Arginina/metabolismo , Linhagem Celular , Cisteína/química , Cisteína/metabolismo , Citoproteção , Glicólise/efeitos dos fármacos , Humanos , Imidazóis/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fator 2 Relacionado a NF-E2/agonistas , Fosfoglicerato Quinase/antagonistas & inibidores , Multimerização Proteica , Aldeído Pirúvico/química , Aldeído Pirúvico/metabolismo , Aldeído Pirúvico/farmacologia , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/genética , Transcrição Gênica , Ubiquitinação
4.
Proc Natl Acad Sci U S A ; 114(46): E9903-E9912, 2017 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-29087350

RESUMO

Expression of the transcription factor FOXC2 is induced and necessary for successful epithelial-mesenchymal transition, a developmental program that when activated in cancer endows cells with metastatic potential and the properties of stem cells. As such, identifying agents that inhibit the growth of FOXC2-transformed cells represents an attractive approach to inhibit chemotherapy resistance and metastatic dissemination. From a high throughput synthetic lethal screen, we identified a small molecule, FiVe1, which selectively and irreversibly inhibits the growth of mesenchymally transformed breast cancer cells and soft tissue sarcomas of diverse histological subtypes. FiVe1 targets the intermediate filament and mesenchymal marker vimentin (VIM) in a mode which promotes VIM disorganization and phosphorylation during metaphase, ultimately leading to mitotic catastrophe, multinucleation, and the loss of stemness. These findings illustrate a previously undescribed mechanism for interrupting faithful mitotic progression and may ultimately inform the design of therapies for a broad range of mesenchymal cancers.


Assuntos
Mitose/efeitos dos fármacos , Sarcoma/metabolismo , Vimentina/metabolismo , Vimentina/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Descoberta de Drogas , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Filamentos Intermediários/metabolismo , Células-Tronco Neoplásicas/patologia , Fosforilação , Sarcoma/patologia , Fatores de Transcrição/efeitos dos fármacos , Vimentina/química
5.
Angew Chem Int Ed Engl ; 55(26): 7520-4, 2016 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-27145250

RESUMO

Chimeric antigen receptor T (CAR-T) cells have demonstrated promising results against hematological malignancies, but have encountered significant challenges in translation to solid tumors. To overcome these hurdles, we have developed a switchable CAR-T cell platform in which the activity of the engineered cell is controlled by dosage of an antibody-based switch. Herein, we apply this approach to Her2-expressing breast cancers by engineering switch molecules through site-specific incorporation of FITC or grafting of a peptide neo-epitope (PNE) into the anti-Her2 antibody trastuzumab (clone 4D5). We demonstrate that both switch formats can be readily optimized to redirect CAR-T cells (specific for the corresponding FITC or PNE) to Her2-expressing tumor cells, and afford dose-titratable activation of CAR-T cells ex vivo and complete clearance of the tumor in rodent xenograft models. This strategy may facilitate the application of immunotherapy to solid tumors by affording comparable efficacy with improved safety owing to switch-based control of the CAR-T response.


Assuntos
Neoplasias da Mama/terapia , Genes de Troca , Imunoterapia , Receptores de Antígenos de Linfócitos T , Animais , Relação Dose-Resposta a Droga , Feminino , Genes de Troca/genética , Xenoenxertos , Humanos , Camundongos , Receptor ErbB-2/efeitos dos fármacos , Receptor ErbB-2/metabolismo
6.
Nature ; 502(7471): 327-332, 2013 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-24107995

RESUMO

Progressive phases of multiple sclerosis are associated with inhibited differentiation of the progenitor cell population that generates the mature oligodendrocytes required for remyelination and disease remission. To identify selective inducers of oligodendrocyte differentiation, we performed an image-based screen for myelin basic protein (MBP) expression using primary rat optic-nerve-derived progenitor cells. Here we show that among the most effective compounds identifed was benztropine, which significantly decreases clinical severity in the experimental autoimmune encephalomyelitis (EAE) model of relapsing-remitting multiple sclerosis when administered alone or in combination with approved immunosuppressive treatments for multiple sclerosis. Evidence from a cuprizone-induced model of demyelination, in vitro and in vivo T-cell assays and EAE adoptive transfer experiments indicated that the observed efficacy of this drug results directly from an enhancement of remyelination rather than immune suppression. Pharmacological studies indicate that benztropine functions by a mechanism that involves direct antagonism of M1 and/or M3 muscarinic receptors. These studies should facilitate the development of effective new therapies for the treatment of multiple sclerosis that complement established immunosuppressive approaches.


Assuntos
Benzotropina/uso terapêutico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Modelos Biológicos , Esclerose Múltipla/tratamento farmacológico , Bainha de Mielina/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Animais , Antiparkinsonianos/farmacologia , Antiparkinsonianos/uso terapêutico , Benzotropina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Técnicas de Cocultura , Cuprizona/farmacologia , Cuprizona/uso terapêutico , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/patologia , Feminino , Cloridrato de Fingolimode , Sistema Imunitário/efeitos dos fármacos , Sistema Imunitário/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/patologia , Proteína Proteolipídica de Mielina/farmacologia , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Nervo Óptico/citologia , Propilenoglicóis/farmacologia , Propilenoglicóis/uso terapêutico , Ratos , Receptor Muscarínico M1/antagonistas & inibidores , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M3/antagonistas & inibidores , Receptor Muscarínico M3/metabolismo , Recidiva , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Esfingosina/uso terapêutico , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos
7.
Proc Natl Acad Sci U S A ; 110(27): E2510-7, 2013 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-23776209

RESUMO

A cell-based phenotypic screen for inhibitors of biofilm formation in mycobacteria identified the small molecule TCA1, which has bactericidal activity against both drug-susceptible and -resistant Mycobacterium tuberculosis (Mtb) and sterilizes Mtb in vitro combined with rifampicin or isoniazid. In addition, TCA1 has bactericidal activity against nonreplicating Mtb in vitro and is efficacious in acute and chronic Mtb infection mouse models both alone and combined with rifampicin or isoniazid. Transcriptional analysis revealed that TCA1 down-regulates genes known to be involved in Mtb persistence. Genetic and affinity-based methods identified decaprenyl-phosphoryl-ß-D-ribofuranose oxidoreductase DprE1 and MoeW, enzymes involved in cell wall and molybdenum cofactor biosynthesis, respectively, as targets responsible for the activity of TCA1. These in vitro and in vivo results indicate that this compound functions by a unique mechanism and suggest that TCA1 may lead to the development of a class of antituberculosis agents.


Assuntos
Antituberculosos/farmacologia , Benzotiazóis/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Tiofenos/farmacologia , Tuberculose Pulmonar/tratamento farmacológico , Oxirredutases do Álcool , Sequência de Aminoácidos , Animais , Antituberculosos/administração & dosagem , Antituberculosos/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Benzotiazóis/administração & dosagem , Benzotiazóis/química , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Carboidratos Epimerases/antagonistas & inibidores , Carboidratos Epimerases/química , Carboidratos Epimerases/genética , Farmacorresistência Bacteriana , Feminino , Genes Bacterianos , Ensaios de Triagem em Larga Escala , Isoniazida/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Oxirredutases/antagonistas & inibidores , Oxirredutases/química , Oxirredutases/genética , Rifampina/administração & dosagem , Tiofenos/administração & dosagem , Tiofenos/química , Tuberculose Pulmonar/microbiologia
8.
Cell ; 153(6): 1379-93, 2013 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-23746848

RESUMO

Some species mount a robust antibody response despite having limited genome-encoded combinatorial diversity potential. Cows are unusual in having exceptionally long CDR H3 loops and few V regions, but the mechanism for creating diversity is not understood. Deep sequencing reveals that ultralong CDR H3s contain a remarkable complexity of cysteines, suggesting that disulfide-bonded minidomains may arise during repertoire development. Indeed, crystal structures of two cow antibodies reveal that these CDR H3s form a very unusual architecture composed of a ß strand "stalk" that supports a structurally diverse, disulfide-bonded "knob" domain. Diversity arises from somatic hypermutation of an ultralong DH with a severe codon bias toward mutation to cysteine. These unusual antibodies can be elicited to recognize defined antigens through the knob domain. Thus, the bovine immune system produces an antibody repertoire composed of ultralong CDR H3s that fold into a diversity of minidomains generated through combinations of somatically generated disulfides.


Assuntos
Diversidade de Anticorpos , Bovinos/imunologia , Regiões Determinantes de Complementaridade , Imunoglobulina G/genética , Imunoglobulina M/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Cisteína/análise , Cisteína/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoglobulina G/química , Imunoglobulina M/química , Camundongos , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Alinhamento de Sequência
9.
Proc Natl Acad Sci U S A ; 110(11): 4261-6, 2013 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-23440204

RESUMO

Somatic hypermutation and clonal selection lead to B cells expressing high-affinity antibodies. Here we show that somatic mutations not only play a critical role in antigen binding, they also affect the thermodynamic stability of the antibody molecule. Somatic mutations directly involved in antigen recognition by antibody 93F3, which binds a relatively small hapten, reduce the melting temperature compared with its germ-line precursor by up to 9 °C. The destabilizing effects of these mutations are compensated by additional somatic mutations located on surface loops distal to the antigen binding site. Similarly, somatic mutations enhance both the affinity and thermodynamic stability of antibody OKT3, which binds the large protein antigen CD3. Analysis of the crystal structures of 93F3 and OKT3 indicates that these somatic mutations modulate antibody stability primarily through the interface of the heavy and light chain variable domains. The historical view of antibody maturation has been that somatic hypermutation and subsequent clonal selection increase antigen-antibody specificity and binding energy. Our results suggest that this process also optimizes protein stability, and that many peripheral mutations that were considered to be neutral are required to offset deleterious effects of mutations that increase affinity. Thus, the immunological evolution of antibodies recapitulates on a much shorter timescale the natural evolution of enzymes in which function and thermodynamic stability are simultaneously enhanced through mutation and selection.


Assuntos
Anticorpos Monoclonais Murinos/imunologia , Afinidade de Anticorpos/fisiologia , Especificidade de Anticorpos/imunologia , Sítios de Ligação de Anticorpos/imunologia , Região Variável de Imunoglobulina/imunologia , Hipermutação Somática de Imunoglobulina/fisiologia , Animais , Anticorpos Monoclonais Murinos/genética , Especificidade de Anticorpos/genética , Sítios de Ligação de Anticorpos/genética , Células HEK293 , Humanos , Região Variável de Imunoglobulina/genética , Camundongos , Mutação , Estabilidade Proteica
10.
Proc Natl Acad Sci U S A ; 108(27): 11052-6, 2011 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-21690365

RESUMO

We report a bacterial system for the evolution of cyclic peptides that makes use of an expanded set of amino acid building blocks. Orthogonal aminoacyl-tRNA synthetase/tRNA(CUA) pairs, together with a split intein system were used to biosynthesize a library of ribosomal peptides containing amino acids with unique structures and reactivities. This peptide library was subsequently used to evolve an inhibitor of HIV protease using a selection based on cellular viability. Two of three cyclic peptides isolated after two rounds of selection contained the keto amino acid p-benzoylphenylalanine (pBzF). The most potent peptide (G12: GIXVSL; X=pBzF) inhibited HIV protease through the formation of a covalent Schiff base adduct of the pBzF residue with the ε-amino group of Lys 14 on the protease. This result suggests that an expanded genetic code can confer an evolutionary advantage in response to selective pressure. Moreover, the combination of natural evolutionary processes with chemically biased building blocks provides another strategy for the generation of biologically active peptides using microbial systems.


Assuntos
Evolução Molecular Direcionada/métodos , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/farmacologia , Inibidores de Proteases/farmacologia , Aminoácidos/química , Sequência de Bases , Primers do DNA/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , Biblioteca de Peptídeos , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/química , Inibidores de Proteases/química
11.
Biochemistry ; 50(11): 1894-900, 2011 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-21280675

RESUMO

We have employed a rapid fluorescence-based screen to assess the polyspecificity of several aminoacyl-tRNA synthetases (aaRSs) against an array of unnatural amino acids. We discovered that a p-cyanophenylalanine specific aminoacyl-tRNA synthetase (pCNF-RS) has high substrate permissivity for unnatural amino acids, while maintaining its ability to discriminate against the 20 canonical amino acids. This orthogonal pCNF-RS, together with its cognate amber nonsense suppressor tRNA, is able to selectively incorporate 18 unnatural amino acids into proteins, including trifluoroketone-, alkynyl-, and halogen-substituted amino acids. In an attempt to improve our understanding of this polyspecificity, the X-ray crystal structure of the aaRS-p-cyanophenylalanine complex was determined. A comparison of this structure with those of other mutant aaRSs showed that both binding site size and other more subtle features control substrate polyspecificity.


Assuntos
Aminoacil-tRNA Sintetases/química , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Nitrilas/química , Nitrilas/metabolismo , Conformação Proteica , RNA de Transferência/genética , RNA de Transferência/metabolismo , Especificidade por Substrato
12.
J Mol Biol ; 395(2): 361-74, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19852970

RESUMO

We report a new vector, pEVOL, for the incorporation of unnatural amino acids into proteins in Escherichia coli using evolved Methanocaldococcus jannaschii aminoacyl-tRNA synthetase(s) (aaRS)/suppressor tRNA pairs. This new system affords higher yields of mutant proteins through the use of both constitutive and inducible promoters to drive the transcription of two copies of the M. jannaschii aaRS gene. Yields were further increased by coupling the dual-aaRS promoter system with a newly optimized suppressor tRNA(CUA)(opt) in a single-vector construct. The optimized suppressor tRNA(CUA)(opt) afforded increased plasmid stability compared with previously reported vectors for unnatural amino acid mutagenesis. To demonstrate the utility of this new system, we introduced 14 mutant aaRS into pEVOL and compared their ability to insert unnatural amino acids in response to three independent amber nonsense codons in sperm whale myoglobin or green fluorescent protein. When cultured in rich media in shake flasks, pEVOL was capable of producing more than 100 mg/L mutant GroEL protein. The versatility, increased yields, and increased stability of the pEVOL vector will further facilitate the expression of proteins with unnatural amino acids.


Assuntos
Aminoácidos/genética , Aminoácidos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos , Aminoácidos/química , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Sequência de Bases , Chaperonina 60/genética , Evolução Molecular Direcionada , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Genes Arqueais , Methanococcales/enzimologia , Methanococcales/genética , Dados de Sequência Molecular , Mutagênese , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação de Ácido Nucleico , Plasmídeos/genética , RNA de Transferência/química , RNA de Transferência/genética
13.
Biochemistry ; 48(12): 2643-53, 2009 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-19265424

RESUMO

To increase the utility of protein mutagenesis with unnatural amino acids, a recombinant expression system in the methylotrophic yeast Pichia pastoris was developed. Aminoacyl-tRNA synthetase/suppressor tRNA (aaRS/tRNA(CUA)) pairs previously evolved in Saccharomyces cerevisiae to be specific for unnatural amino acids were inserted between eukaryotic transcriptional control elements and stably incorporated into the P. pastoris genome. Both the Escherichia coli tyrosyl- and leucyl-RS/tRNA(CUA) pairs were shown to be orthogonal in P. pastoris and used to incorporate eight unnatural amino acids in response to an amber codon with high yields and fidelities. In one example, we show that a recombinant human serum albumin mutant containing a keto amino acid (p-acetylphenylalanine) can be efficiently expressed in this system and selectively conjugated via oxime ligation to a therapeutic peptide mimetic containing an permittivity-(2-(aminooxy)acetyl)-L-lysine residue. Moreover, unnatural amino acid expression in the methylotrophic host was systematically optimized by modulation of aaRS levels to express mutant human serum albumin in excess of 150 mg/L in shake flasks, more than an order of magnitude better than that reported in S. cerevisiae. This methodology should allow the production of high yields of complex proteins containing unnatural amino acids whose expression is not practical in existing systems.


Assuntos
Pichia/enzimologia , Pichia/genética , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Espectrometria de Massas , Fenótipo , Pichia/metabolismo , Engenharia de Proteínas , RNA de Transferência/metabolismo
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