RESUMO
BACKGROUND: Human lysozyme (hLYZ) is a natural antibacterial protein with broad applications in food and pharmaceutical industries. Recombinant production of hLYZ in Komagataella phaffii (K. phaffii) has attracted considerable attention, but there are very limited strategies for its hyper-production in yeast. RESULTS: Here through Atmospheric and Room Temperature Plasma (ARTP)-based mutagenesis and transcriptomic analysis, the expression of two genes MYO1 and IQG1 encoding the cytokinesis core proteins was identified downregulated along with higher hLYZ production. Deletion of either gene caused severe cytokinesis defects, but significantly enhanced hLYZ production. The highest hLYZ yield of 1,052,444 ± 23,667 U/mL bioactivity and 4.12 ± 0.11 g/L total protein concentration were obtained after high-density fed-batch fermentation in the Δmyo1 mutant, representing the best production of hLYZ in yeast. Furthermore, O-linked mannose glycans were characterized on this recombinant hLYZ. CONCLUSIONS: Our work suggests that cytokinesis-based morphology engineering is an effective way to enhance the production of hLYZ in K. phaffii.
Assuntos
Muramidase , Proteínas Recombinantes , Saccharomycetales , Muramidase/metabolismo , Muramidase/genética , Muramidase/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales/metabolismo , Saccharomycetales/genética , Humanos , Fermentação , Citocinese , Engenharia Metabólica/métodos , Técnicas de Cultura Celular por LotesRESUMO
Mycotoxins have substantial impacts on agricultural production and food preservation. Some have high similarities in bioactivity but subtle differences on structures from various fungal producers. Understanding of their complex cross-biosynthesis will provide new insights into enzyme functions and food safety. Here, based on structurally related mycotoxins, such as aurovertins, asteltoxin, and citreoviridin, we showed that methyltransferase (MT)-catalyzed methylation is required for efficient oxidation and polyketide stability. MTs have broad interactions with polyketide synthases and flavin-containing monooxygenases (FMOs), while MT AstB is required for FMO AstC functionality in vivo. FMOs have common catalysis on pyrone-polyene intermediates but different catalytic specificity and efficiency on oxidative intermediates for the selective production of more toxic and complex mycotoxins. Thus, the subtle protein interaction and elaborate versatile catalysis of biosynthetic enzymes contribute to the efficient and selective biosynthesis of these structure-related mycotoxins and provide the basis to re-evaluate and control mycotoxins for agricultural and food safety.
Assuntos
Micotoxinas , Policetídeos , Micotoxinas/química , Policetídeos/metabolismo , Metiltransferases , Policetídeo Sintases/metabolismo , CatáliseRESUMO
A method was developed for the determination of N,N'-ethylenediamine disuccinic acid (EDDS) in bioconversion samples by high performance liquid chromatography (HPLC). An InertSustain AQ-C18 column (250 mm×4.6 mm, 5 µm) was used in the analysis. The mobile phase was 25% (v/v) methanol with 1.0 g/L Cu(CO2CH3)2·H2O, 2.0 g/L tetrabutylammonium hydroxide, and the pH was adjusted to 2.80 with phosphoric acid. The flow rate of the mobile phase was 1.0 mL/min, and the column temperature was set at 30â. The detection wavelength was 254 nm. EDDS, fumaric acid, citric acid, malic acid and ethylenediaminetetraacetic acid (EDTA) were separated from one another within 8 min. EDDS showed good linearity in the range of 0.06-0.6 g/L. About 0.25 g/L EDDS was detected in the biosynthesis reaction solution catalyzed by recombinant EDDS-lyase, while 36.56 g/L malic acid was formed as the by-product from fumaric acid. The catalytic activity of the enzyme was confirmed in the hydrolysis of 10 g/L EDDS, which produced 3.05 g/L malic acid in 3 h. This analytical method is simple, rapid, sensitive, reliable, and suitable for the analysis in the research of EDDS bioconversion process.
Assuntos
Cromatografia Líquida de Alta Pressão , Etilenodiaminas/análise , Succinatos/análiseRESUMO
Paired box protein 5 (PAX5) plays a lineage determination role in B-cell development. However, high expression of PAX5 has been also found in various malignant diseases, including B-lymphoproliferative disorders (B-LPDs), but its functions and mechanisms in these diseases are still unclear. Here, we show that PAX5 induces drug resistance through association and activation of receptor-interacting serine/threonine-protein kinase 2 (RIP2; also known as RIPK2), and subsequent activation of NF-κB signaling and anti-apoptosis gene expression in B-lymphoproliferative cells. Furthermore, PAX5 is able to interact with RIP1 and RIP3, modulating both RIP1-mediated TNFR and RIP2-mediated NOD1 and NOD2 pathways. Our findings describe a new function of PAX5 in regulating RIP1 and RIP2 activation, which is at least involved in chemotherapeutic drug resistance in B-LPDs.
Assuntos
Linfócitos B/metabolismo , Resistencia a Medicamentos Antineoplásicos , Transtornos Linfoproliferativos/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição PAX5/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linfócitos B/efeitos dos fármacos , Bortezomib/farmacologia , Bortezomib/uso terapêutico , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Transtornos Linfoproliferativos/patologia , Modelos Biológicos , Proteínas Adaptadoras de Sinalização NOD/metabolismo , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Agkisacucetin extracted from the venom of Agkistrodon acutus has been demonstrated to be a promising antithrombotic drug candidate in clinical studies due to its function as a novel platelet membrane glycoprotein (GP) Ib inhibitor. Agkisacucetin is a heterodimeric protein composed of α- and ß-subunits with seven disulphide bonds. Both subunits form inactive homodimeric products, which cause difficulties for recombinant production. In this study, Agkisacucetin α- and ß-subunits were inserted sequentially into the chromosome of Pichia pastoris at the mutant histidinol dehydrogenase gene and ribosomal DNA repeat sites, respectively. By optimizing the gene copies and productivity of each subunit by drug screening, we successfully obtained a recombinant strain with balanced expression of the two subunits. Using this strain, a yield greater than 100 mg/L recombinant Agkisacucetin in fed-batch fermentation was reached. The recombinant Agkisacucetin possessed extremely similar binding affinity to recombinant GPIb and human platelets in in vitro assays, and its ristocetin-induced platelet aggregation activity ex vivo was identical to that of the extracted native Agkisacucetin, demonstrating that the yeast-derived Agkisacucetin could be an effective alternative to native Agkisacucetin. Moreover, this study provides an effective strategy for balancing the expression and production of heterodimeric proteins in P. pastoris.
Assuntos
Antineoplásicos/metabolismo , Venenos de Crotalídeos/metabolismo , Agkistrodon/metabolismo , Sequência de Aminoácidos , Animais , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Técnicas de Cultura Celular por Lotes , Venenos de Crotalídeos/química , Venenos de Crotalídeos/genética , Dimerização , Humanos , Dados de Sequência Molecular , Pichia/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIb-IX de Plaquetas/antagonistas & inibidores , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Ristocetina/farmacologiaRESUMO
BACKGROUND: The yeast Pichia pastoris (P. pastoris) has become a popular 'cell factory' for producing heterologous proteins, but production widely varies among proteins. Cultivation temperature is frequently reported to significantly affect protein production; however, the underlying mechanisms of this effect remain unclear. RESULTS: A P. pastoris strain expressing recombinant human interleukin-10 (rhIL-10) under the control of the AOX1 promoter was used as the model in this study. This system shows high-yield rhIL-10 production with prolonged methanol-induction times when cultured at 20°C but low-yield rhIL-10 production and higher cell death rates when cultured at 30°C. Further investigation showed that G3-pro-rhIL10, an immature form of rhIL-10 that contains the glycosylation-modified signal peptide, remained in the ER for a prolonged period at 30°C. The retention resulted in higher ER stress levels that were accompanied by increased ROS production, Ca(2+) leakage, ER-containing autophagosomes, shortened cortical ER length and compromised induction of the unfolded protein response (UPR). In contrast, G3-pro-rhIL10 was quickly processed and eliminated from the ER at 20°C, resulting in a lower level of ER stress and improved rhIL-10 production. CONCLUSIONS: High-temperature cultivation of an rhIL-10 expression strain leads to prolonged retention of immature G3-pro-rhIL10 in ER, causing higher ER stress levels and thus greater yeast cell death rates and lower production of rhIL-10.
Assuntos
Estresse do Retículo Endoplasmático , Interleucina-10/biossíntese , Pichia/metabolismo , Regiões Promotoras Genéticas , Resposta a Proteínas não Dobradas , Temperatura Alta , Humanos , Interleucina-10/genética , Pichia/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
CD83 is a highly glycosylated type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. CD83 is upregulated during dendritic cell (DC) maturation, which is critical for the initiation of adaptive immune responses. The soluble isoform of CD83 (sCD83) is encoded by alternative splicing from full-length CD83 mRNA and inhibits DC maturation, which suggests that sCD83 acts as a potential immune suppressor. In this study, we developed a sound strategy to express functional sCD83 from Pichia pastoris in extremely high-density fermentation. Purified sCD83 was expressed as a monomer at a yield of more than 200 mg/L and contained N-linked glycosylation sites that were characterized by PNGase F digestion. In vitro tests indicated that recombinant sCD83 bound to its putative counterpart on monocytes and specifically blocked the binding of anti-CD83 antibodies to cell surface CD83 on DCs. Moreover, sCD83 from yeast significantly suppressed ConA-stimulated PBMC proliferation. Therefore, sCD83 that was expressed from the P. pastoris was functionally active and may be used for in vivo and in vitro studies as well as future clinical applications.
Assuntos
Antígenos CD/metabolismo , Células Dendríticas/citologia , Fermentação , Imunoglobulinas/metabolismo , Leucócitos Mononucleares/citologia , Glicoproteínas de Membrana/metabolismo , Monócitos/citologia , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Antígenos CD/genética , Antígenos CD/isolamento & purificação , Western Blotting , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Células Dendríticas/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoglobulinas/genética , Imunoglobulinas/isolamento & purificação , Leucócitos Mononucleares/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Monócitos/metabolismo , Pichia/crescimento & desenvolvimento , Plasmídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Antígeno CD83RESUMO
Granulysin is a cytolytic, proinflammatory protein produced by human cytolytic T-lymphocytes and natural killer cells. Granulysin has two stable isoforms with molecular weight of 9 and 15 kDa; the 9-kDa form is a result of proteolytic maturation of the 15-kDa precursor. Recombinant 9-kDa granulysin exhibits cytolytic activity against a variety of microbes, such as bacteria, parasites, fungi, yeast and a variety of tumor cell lines. However, it is difficult to produce granulysin in large quantities by traditional methods. In this study, we developed a simple and robust fed-batch fermentation process for production and purification of recombinant 9- and 15-kDa granulysin using Pichia pastoris in a basal salt medium at high cell density. The granulysin yield reaches at least 100 mg/l in fermentation, and over 95 % purity was achieved with common His-select affinity and ion exchange chromatography. Functional analysis revealed that the yeast-expressed granulysin displayed dose-dependent target cytotoxicity. These results suggest that fermentation in P. pastoris provides a sound strategy for large-scale recombinant granulysin production that may be used in clinical applications and basic research.
Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Técnicas de Cultura Celular por Lotes/métodos , Pichia/metabolismo , Antígenos de Diferenciação de Linfócitos T/química , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/isolamento & purificação , Reatores Biológicos/microbiologia , Fermentação , Peso Molecular , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Interleukin (IL)-10 is an anti-inflammatory cytokine that could be potentially applied for clinical therapy. However, its short circulating half-life in the serum limits its clinical applications. In this study, we designed a fusion protein containing human IL-10 and an IgG Fc fragment (hIL-10/Fc), and expressed it in Pichia pastoris. This hIL-10/Fc fusion protein was purified from the culture supernatant using MabSelect affinity chromatography and size-exclusion chromatography. The hIL-10/Fc yield was about 5mg/L in shake flasks, with purity exceeding 95%. In addition, the hIL-10/Fc fusion protein suppressed the phytohemagglutinin-induced IFN-γ production in human peripheral blood mononuclear cells. Pharmacokinetic study also revealed that hIL-10/Fc has a prolonged circulating half-life of about 30h in rats. More importantly, the hIL-10/Fc fusion protein displayed highly specific biological activity, which was slightly higher than that of the commercial recombinant human IL-10 (rhIL-10). Therefore, P. pastoris is useful in the large-scale production of hIL-10/Fc fusion protein for both research and therapeutic applications.