RESUMO
Komagataella phaffii, also known as Pichia pastoris, is a common host for the production of biologics and enzymes, due to fast growth, high productivity, and advancements in host engineering. Several K. phaffii variants are commonly used as interchangeable base strains, which confounds efforts to improve this host. In this study, genomic and transcriptomic analyses of Y-11430 (CBS7435), GS115, X-33, and eight other variants enabled a comparative assessment of the relative fitness of these hosts for recombinant protein expression. Cell wall integrity explained the majority of the variation among strains, impacting transformation efficiency, growth, methanol metabolism, and secretion of heterologous proteins. Y-11430 exhibited the highest activity of genes involved in methanol utilization, up to two-fold higher transcription of heterologous genes, and robust growth. With a more permeable cell wall, X-33 displayed a six-fold higher transformation efficiency and up to 1.2-fold higher titers than Y-11430. X-33 also shared nearly all mutations, and a defective variant of HIS4, with GS115, precluding robust growth. Transferring two beneficial mutations identified in X-33 into Y-11430 resulted in an optimized base strain that provided up to four-fold higher transformation efficiency and three-fold higher protein titers, while retaining robust growth. The approach employed here to assess unique banked variants in a species and then transfer key beneficial variants into a base strain should also facilitate rational assessment of a broad set of other recombinant hosts.
Assuntos
Proteínas Fúngicas/genética , Genoma Fúngico/genética , Pichia/genética , Proteínas Recombinantes/genética , Transcriptoma/genética , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Genômica , Pichia/metabolismo , RNA Fúngico/análise , RNA Fúngico/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Análise de Sequência de RNARESUMO
E3 ubiquitin ligases catalyze the final step of ubiquitin conjugation and regulate numerous cellular processes. The HECT class of E3 ubiquitin (Ub) ligases directly transfers Ub from bound E2 enzyme to a myriad of substrates. The catalytic domain of HECT Ub ligases has a bilobal architecture that separates the E2 binding region and catalytic site. An important question regarding HECT domain function is the control of ligase activity and specificity. Here we present a functional analysis of the HECT domain of the E3 ligase HUWE1 based on crystal structures and show that a single N-terminal helix significantly stabilizes the HECT domain. We observe that this element modulates HECT domain activity, as measured by self-ubiquitination induced in the absence of this helix, as distinct from its effects on Ub conjugation of substrate Mcl-1. Such subtle changes to the protein may be at the heart of the vast spectrum of substrate specificities displayed by HECT domain E3 ligases.
Assuntos
Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Domínio Catalítico/fisiologia , Cristalografia por Raios X , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Estrutura Secundária de Proteína/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Especificidade por Substrato/fisiologia , Proteínas Supressoras de Tumor , Ubiquitina/química , Ubiquitina/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genéticaRESUMO
We present here a new method to enhance the detection of secreted cytokines and chemokines from single human mononuclear cells. The technique uses a hybridization chain reaction (HCR) to amplify signals resulting from sandwich immunoassays. This immuno-HCR employs oligonucleotide-based initiators covalently linked to antibodies to propagate a chain reaction of hybridization events involving a pair of complementary hairpin oligomers bearing fluorescent labels. Integrating this strategy for signal amplification with microengraving (a soft lithographic method for printing arrays of secreted proteins from thousands of single cells) improves both the limits of detection and sensitivity for cytokines and chemokines captured from individual cells by an average of 200-fold relative to methods for direct detection by fluoresence. This approach should enhance the utility of microengraving for defining the immunological signatures of diseases and responses to interventional therapies based on multiplexed single-cell analysis.
Assuntos
Citocinas/análise , Imunoensaio/métodos , Leucócitos Mononucleares/metabolismo , Anticorpos Imobilizados/imunologia , Células Cultivadas , Quimiocinas/análise , Quimiocinas/imunologia , Citocinas/imunologia , Dimetilpolisiloxanos/química , Corantes Fluorescentes/química , Vidro/química , Humanos , Leucócitos Mononucleares/imunologia , Oligonucleotídeos/química , Análise Serial de Proteínas/métodosRESUMO
The selection of highly productive cell lines remains a key step for manufacturing therapeutic proteins. Microengraving was used to screen chemically mutagenized populations of Pichia pastoris for increased production of an Fc fragment. Clones retrieved following three rounds of mutagenesis yielded titers 2.65-fold greater than those of the parental strain.
Assuntos
Biotecnologia/métodos , Fragmentos Fc das Imunoglobulinas/metabolismo , Pichia/metabolismo , Bioensaio/métodos , Fragmentos Fc das Imunoglobulinas/genética , Programas de Rastreamento/métodos , Mutagênese , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The production of heterologous proteins by secretion from cellular hosts is an important determinant for the cost of biotherapeutics. A single-cell analytical method called microengraving was used to examine the heterogeneity in secretion by the methylotrophic yeast Pichia pastoris. We show that constitutive secretion of a human Fc fragment by P. pastoris is not cell-cycle dependent, but rather fluctuates between states of high and low productivity in a stochastic manner.
Assuntos
Técnicas de Cultura de Células/instrumentação , Perfilação da Expressão Gênica/instrumentação , Modelos Biológicos , Pichia/fisiologia , Análise Serial de Proteínas/instrumentação , Proteínas Recombinantes/metabolismo , Simulação por Computador , Desenho de Equipamento , Processos Estocásticos , Integração de SistemasRESUMO
Constructing efficient cellular factories often requires integration of heterologous pathways for synthesis of novel compounds and improved cellular productivity. Few genomic sites are routinely used, however, for efficient integration and expression of heterologous genes, especially in nonmodel hosts. Here, a data-guided framework for informing suitable integration sites for heterologous genes based on ATAC-seq was developed in the nonmodel yeast Komagataella phaffii. Single-copy GFP constructs were integrated using CRISPR/Cas9 into 38 intergenic regions (IGRs) to evaluate the effects of IGR size, intensity of ATAC-seq peaks, and orientation and expression of adjacent genes. Only the intensity of accessibility peaks was observed to have a significant effect, with higher expression observed from IGRs with low- to moderate-intensity peaks than from high-intensity peaks. This effect diminished for tandem, multicopy integrations, suggesting that the additional copies of exogenous sequence buffered the transcriptional unit of the transgene against effects from endogenous sequence context. The approach developed from these results should provide a basis for nominating suitable IGRs in other eukaryotic hosts from an annotated genome and ATAC-seq data.
Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , DNA Intergênico/genética , Sistemas CRISPR-Cas/genética , Edição de Genes , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/metabolismo , Hormônio do Crescimento Humano/genética , Hormônio do Crescimento Humano/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismoRESUMO
The addition of ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers to proteins serves to modulate function and is a key step in protein degradation, epigenetic modification and intracellular localization. Deubiquitinating enzymes and Ubl-specific proteases, the proteins responsible for the removal of Ub and Ubls, act as an additional level of control over the ubiquitin-proteasome system. Their conservation and widespread occurrence in eukaryotes, prokaryotes and viruses shows that these proteases constitute an essential class of enzymes. Here, we discuss how chemical tools, including activity-based probes and suicide inhibitors, have enabled (i) discovery of deubiquitinating enzymes, (ii) their functional profiling, crystallographic characterization and mechanistic classification and (iii) development of molecules for therapeutic purposes.
Assuntos
Biologia , Endopeptidases/metabolismo , Inibidores Enzimáticos/farmacologia , Animais , Endopeptidases/química , Endopeptidases/genética , Ativação Enzimática/efeitos dos fármacos , Humanos , Cinética , Filogenia , Proteases Específicas de UbiquitinaRESUMO
Biologic drugs are promoting growth in the biopharmaceutical industry. Despite the clinical benefits of these drugs, the time and costs required to bring new biologics to market still are substantial. Three key challenges, among others, persist in the development of biologic drugs: namely, establishing product similarity, product toxicity, and global accessibility. New classes of microtools that facilitate the isolation and interrogation of single cells have the potential to impact each of these challenges. This opinion considers recent examples of microtools with demonstrated or potential utility to address problems in these areas. Integrating these advanced technologies into the development of new biologics could greatly reduce time and costs required to bring alternative products to market, and thus expand their global availability.
Assuntos
Biofarmácia/métodos , Biotecnologia/métodos , Microtecnologia/métodos , Análise de Célula Única/métodos , Animais , HumanosRESUMO
Biopharmaceuticals represent the fastest growing sector of the global pharmaceutical industry. Cost-efficient production of these biologic drugs requires a robust host organism for generating high titers of protein during fermentation. Understanding key cellular processes that limit protein production and secretion is, therefore, essential for rational strain engineering. Here, with single-cell resolution, we systematically analysed the productivity of a series of Pichia pastoris strains that produce different proteins both constitutively and inducibly. We characterized each strain by qPCR, RT-qPCR, microengraving, and imaging cytometry. We then developed a simple mathematical model describing the flux of folded protein through the ER. This combination of single-cell measurements and computational modelling shows that protein trafficking through the secretory machinery is often the rate-limiting step in single-cell production, and strategies to enhance the overall capacity of protein secretion within hosts for the production of heterologous proteins may improve productivity.
Assuntos
Pichia/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Via Secretória , Análise de Célula Única/métodos , Algoritmos , Retículo Endoplasmático/metabolismo , Citometria de Fluxo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Pichia/genética , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transformação GenéticaRESUMO
Protein modification by ubiquitin (Ub) and ubiquitin-like modifiers (Ubl) requires the action of activating (E1), conjugating (E2), and ligating (E3) enzymes and is a key step in the specific destruction of proteins. Deubiquitinating enzymes (DUBs) deconjugate substrates modified with Ub/Ubl's and recycle Ub inside the cell. Genome mining based on sequence homology to proteins with known function has assigned many enzymes to this pathway without confirmation of either conjugating or DUB activity. Function-dependent methodologies are still the most useful for rapid identification or assessment of biological activity of expressed proteins from cells. Activity-based protein profiling uses chemical probes that are active-site-directed for the classification of protein activities in complex mixtures. Here we show that the design and use of an expanded set of Ub-based electrophilic probes allowed us to recover and identify members of each enzyme class in the ubiquitin-proteasome system, including E3 ligases and DUBs with previously unverified activity. We show that epitope-tagged Ub-electrophilic probes can be used as activity-based probes for E3 ligase identification by in vitro labeling and activity studies of purified enzymes identified from complex mixtures in cell lysate. Furthermore, the reactivity of our probe with the HECT domain of the E3 Ub ligase ARF-BP1 suggests that multiple cysteines may be in the vicinity of the E2-binding site and are capable of the transfer of Ub to self or to a substrate protein.
Assuntos
Sondas Moleculares/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina/metabolismo , Sequência de Aminoácidos , Animais , Camundongos , Sondas Moleculares/síntese química , Sondas Moleculares/química , Dados de Sequência Molecular , Estrutura Molecular , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Alinhamento de Sequência , Ubiquitina/química , Complexos Ubiquitina-Proteína Ligase/genéticaRESUMO
Glycosyl phosphate and trichloroacetimidate monosaccharide building blocks were used in a stepwise solution-phase synthesis of three Lewis blood group oligosaccharides. The syntheses were conducted to establish general routes for the automated assembly of the oligosaccharide portion of biologically important glycolipids. The H-type II pentasaccharide, Le(x) pentasaccharide, and Le(y) hexasaccharide were prepared in high yield. These syntheses served to evaluate the utility and limitations of the 2-(azidomethyl)benzoate ester (AZMB) for the construction of complex carbohydrates. Development of a glucosamine building block containing a N-trichloroacetamide group to mask the C2 amine improved coupling yields and was key for completion of the Le(x) and Le(y) structures.