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Alpha-amilase de Acanthoscelides obtectus: clonagem e expressão heteróloga em Pichia pastoris. Repositório Alice
ÁTILA-SANTOS, D.; MULINARI, F.; CHRISPEELS, M. J.; GROSSI-DE-SÁ, M. F..
2006
Tipo: Resumo em anais de congresso (ALICE) Palavras-chave: Biologia molecular; Acanthoscelides obtectus; Alpha-amilase; Pichia pastoris; Clonagem; Expressão heteróloga.
Ano: 2006 URL: http://www.alice.cnptia.embrapa.br/handle/doc/188522
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Atividade fungistática de uma quitinase recombinante do feijão de corda [vigna unguiculata (l.) (walp.)] Contra lasiodiplodia theobromae pat. (griff. e maubl.), agente causal da resinose do cajueiro (anacardium occidentale l.) Repositório Alice
LOPES NETO, A. V..
O objetivo deste trabalho foi avaliar a atividade biológica de uma quitinase recombinante (rVuChi) de feijão-caupi (Vigna unguiculata) contra o fungo fitopatogênico Lasiodiplodia theobromae. A proteína recombinante foi expressa em Pichia pastoris, coletada e purificada após 72h de indução, utilizando cromatografia de afinidade em matriz de quitina. A quitinase foi eluída a partir da cromatografia de afinidade com ácido acético a 0,1 M. Ensaio enzimático foi realizado contra o substrato sintético (quitina coloidal), a fim de determinar a atividade da proteína recombinante purificada. A quitinase apresentou atividade específica de 5.637,32 U/mg de proteína. Testes biológicos foram realizados. Nestes testes três diferentes isolados de L. theobromae,...
Tipo: Tese/dissertação (ALICE) Palavras-chave: RVuChi; Fungo fitopatogênico; Pichia pastoris.
Ano: 2014 URL: http://www.alice.cnptia.embrapa.br/alice/handle/doc/1102106
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Avaliação de métodos para obtenção de proteínas recombinantes. Repositório Alice
SANTOS JUNIOR, A. G. dos; PIRAINE, R. E. A.; CUNHA, R. C.; NIZOLI, L. Q.; ANDREOTTI, R.; LEITE, F. P. L..
A utilização de Pichia pastoris para a produção de proteínas recombinantes apresenta inúmeras vantagens que esse sistema agrega, destacando-se a exportação das moléculas para o meio extracelular. Isto permite que a obtenção das proteínas possa ser realizada por métodos de precipitação, com uso de sais neutros ou solventes orgânicos, sem trazer prejuízos. O objetivo deste trabalho foi avaliar a utilização de sulfato de amônio, acetona e metanol para obtenção da proteína recombinante Bm86 do carrapato Rhipicephalus microplus expressa por P. pastoris. A ação provocada pelos solventes orgânicos ocasionou uma solubilidade parcial das proteínas. Na quantificação por densitometria da proteína rBm86-CG, verificou-se que utilizando acetona (1:2) foi obtido 30,3...
Tipo: Artigo em periódico indexado (ALICE) Palavras-chave: Sais neutros; Antigenicidade; Solvente orgânico; Pichia pastoris; Rhipicephalus microplus.
Ano: 2017 URL: http://www.alice.cnptia.embrapa.br/alice/handle/doc/1082504
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Clonagem e expressão de genes de monoxigenases de polissacarídeos líticas (LPMOs) de fungos em Pichia pastoris. Repositório Alice
SANTANA, M. W. P. R. de.; RODRIGUES, K. B.; SANTOS, F. P. dos.; MENDES, T. D.; DAMASO, M. C. T.; FAVARO, L. C. de L.; SIQUEIRA, F. G. de.
2016
Tipo: Artigo em anais de congresso (ALICE) Palavras-chave: Seleção de genes candidatos; Genes candidatos de fungos; LPMOs de fungos; Clonagem de genes; Expressão de genes; Pichia pastoris.
Ano: 2016 URL: http://www.alice.cnptia.embrapa.br/handle/doc/1056933
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Comparing the expression of human DNA topoisomerase I in KM71H and X33 strains of Pichia pastoris Electron. J. Biotechnol.
Ping,Ruo; Sin Teoh,Leong; Kwai Chan,Mooi; Miswan,Noorizan; Yin Khoo,Boon.
Background: Human is an essential cellular enzyme that is found in all human cells. As this enzyme is upregulated in cancer cells exceedingly, it is used as a target for cancer chemotherapeutic drug development. As such, producing the in-house enzyme for the purpose to speed up the search for more cost-effective and target specific hTopoI inhibitors is warranted. This study aims to compare the optimised conditions for the expression of hTopoI in KM71H (MutS) and X33 (Mut+) strains of Pichia pastoris. P. pastoris transfected with an hTopoI recombinant vector was used for the optimization of a higher level of hTopoI expression. Results: In the process, fed-batch cultivation parameters that influence the expression of hTopoI, such as culture temperature,...
Tipo: Journal article Palavras-chave: Cell density; Pichia pastoris; Total protein concentration; Recombinant protein expression.
Ano: 2016 URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000300001
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Construction and expression of two-copy engineered yeast of feruloyl esterase Electron. J. Biotechnol.
Zhou,Min; Huang,Zhiqing; Zhou,Bo; Luo,Yanliu; Jia,Gang; Liu,Guangmang; Zhao,Hua; Chen,Xiaoling.
Background Aspergillus niger has the ability to secrete feruloyl esterase. However, for economically viable industrial applications, it is necessary to increase their catalytic activities and/or protein yields to satisfy the increasing needs for feruloyl esterases. Results The gene AnFaeA that encodes a type A feruloyl esterase was successfully expressed in Pichia pastoris by a two-copy engineered yeast. After a screen in shaker flask, a one-copy strain GSKFA3 having the highest feruloyl esterase activity of 2.4 U/mL was obtained. Then, the pPICZaA-AnFaeA plasmid was transformed into GSKFA3 and the transformants were grown on YPDS plates with antibiotic Zeocin. After cultivation, a two-copy strain GSKZaFA20 with the highest feruloyl esterase activity of...
Tipo: Journal article Palavras-chave: Aspergillus niger; Feruloyl esterase; Pichia pastoris.
Ano: 2015 URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582015000500002
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Construction of an engineered alpha 1-antitrypsin with inhibitory activity based on theoretical studies Electron. J. Biotechnol.
Sangachini,Elham Dasi; Hasannia,Sadegh; Taghdir,Majid; Pirooznia,Nazanin; Ghadicholaei,Kamran Khalili.
Background: The elastase inhibitor α-1-antitrypsin (AAT), is a member of the serpin superfamily of protease inhibitors. AAT has a characteristic secondary structure of three-β-sheets, nine-α-helices and a reactive central loop (RCL). This protein inhibits target proteases by forming a stable complex in which the cleaved RCL is inserted into β-sheet-A of the serpin, leading to a conformational change in the AAT protein. Spontaneous polymerization and instability of AAT are challenges with regard to producing drugs against AAT-deficient diseases. Therefore, the purpose of many investigations currently is to produce drugs with lower degrees of polymerization and higher stabilities. In order to investigate the effect of the...
Tipo: Journal article Palavras-chave: Alpha1-antitrypsin; Molecular dynamic simulation; Pichia pastoris.
Ano: 2012 URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582012000200008
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Cultivation of Pichia pastoris carrying the scFv anti LDL (-) antibody fragment. Effect of preculture carbon source BJM
Arias,Cesar Andres Diaz; Marques,Daniela de Araujo Viana; Malpiedi,Luciana Pellegrini; Maranhão,Andrea Queiroz; Parra,Dulcineia Abdalla Saes; Converti,Attilio; Pessoa Junior,Adalberto.
Abstract Antibodies and antibody fragments are nowadays among the most important biotechnological products, and Pichia pastoris is one of the most important vectors to produce them as well as other recombinant proteins. The conditions to effectively cultivate a P. pastoris strain previously genetically modified to produce the single-chain variable fragment anti low density lipoprotein (-) under the control of the alcohol oxidase promoter have been investigated in this study. In particular, it was evaluated if, and eventually how, the carbon source (glucose or glycerol) used in the preculture preceding cryopreservation in 20% glycerol influences both cell and antibody fragment productions either in flasks or in bioreactor. Although in flasks the volumetric...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Pichia pastoris; ScFv antibody fragment; Cryopreservation; Expression; Carbon source.
Ano: 2017 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822017000300419
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Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastoris Electron. J. Biotechnol.
Arjmand,Sareh; Lotfi,Abbas Sahebghadam; Shamsara,Mehdi; Mowla,Seyed Javad.
Background: Human alpha 1-antitrypsin (AAT) is a potent inhibitor of multiple serine proteases, and protects tissues against their harmful effects. Individuals with reduced or abnormal production of this inhibitor need intravenous administration of exogenous protein. In this study, we employed the methylotrophic (methanol utilizing) yeast Pichia pastoris (P. pastoris) as a preferential host for efficient production and secretion of recombinant AAT. Furthermore, we examined different strategies to maximize the yield of the secreted protein. Results: Our findings revealed that optimizing the codon usage of AAT gene for P. pastoris had positive effects on the level of secreted AAT under the control of inducible alcohol oxidase 1 (AOX1) and constitutive...
Tipo: Journal article Palavras-chave: Human alpha 1-antitrypsin; Pichia pastoris; Recombinant proteins.
Ano: 2013 URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582013000100004
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Engenharia metabólica de Pichia pastoris para produção de ácido glicérico. Repositório Alice
RAMOS, T. G. S.; JUSTEN, F.; ALMEIDA, J. R. M. de.
2015
Tipo: Resumo em anais de congresso (ALICE) Palavras-chave: Pichia pastoris; Ácido glicérico.
Ano: 2015 URL: http://www.alice.cnptia.embrapa.br/handle/doc/1031502
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Engenharia metabólica de Pichia pastoris para produção de ácido xilônico. Repositório Alice
JUSTEN, F.; RAMOS, T. G. S.; ALMEIDA, J. R. M. de.
2015
Tipo: Resumo em anais de congresso (ALICE) Palavras-chave: Pichia pastoris; Ácido xilônico.
Ano: 2015 URL: http://www.alice.cnptia.embrapa.br/handle/doc/1031508
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Expressão heteróloga de monoxigenases de polissacarídeos bacterianas em Pichia pastoris. Repositório Alice
SANTOS, F. P. dos.; RODRIGUES, K. B.; SANTANA, M. W. P. R. de.; SALUM, T. F. C.; FAVARO, L. C. de L.; BRASIL, B. dos S. A. F..
2016
Tipo: Artigo em anais de congresso (ALICE) Palavras-chave: Expressão de LPMOs; Origem bacteriana; Expressão da levedura; Pichia pastoris.
Ano: 2016 URL: http://www.alice.cnptia.embrapa.br/handle/doc/1056928
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Expression and analysis of the glycosylation properties of recombinant human erythropoietin expressed in Pichia pastoris Genet. Mol. Biol.
Teh,Ser Huy; Fong,Mun Yik; Mohamed,Zulqarnain.
The Pichia pastoris expression system was used to produce recombinant human erythropoietin, a protein synthesized by the adult kidney and responsible for the regulation of red blood cell production. The entire recombinant human erythropoietin (rhEPO) gene was constructed using the Splicing by Overlap Extension by PCR (SOE-PCR) technique, cloned and expressed through the secretory pathway of the Pichia expression system. Recombinant erythropoietin was successfully expressed in P. pastoris. The estimated molecular mass of the expressed protein ranged from 32 kDa to 75 kDa, with the variation in size being attributed to the presence of rhEPO glycosylation analogs. A crude functional analysis of the soluble proteins showed that all of the forms were active in...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Erythropoietin; Glycosylation; Pichia pastoris; SOE-PCR.
Ano: 2011 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572011000300017
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High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization BJMBR
Craveiro,R.B.; Ramalho,J.D.; Chagas,J.R.; Wang,P.H.M.; Casarini,D.E.; Pesquero,J.L.; Araújo,R.C.; Pesquero,J.B..
Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Kinins; Human carboxypeptidase M; Recombinant protein; Pichia pastoris.
Ano: 2006 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2006000200007
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Molecular characterization of a novel thermostable laccase PPLCC2 from the brown rot fungus Postia placenta MAD-698-R Electron. J. Biotechnol.
An,Hongde; Xiao,Tingting; Fan,Huan; Wei,Dongsheng.
Background Laccase has been considered important for the degradation of lignocellulose by wood rot fungi. The properties and functions of laccase in white rot fungi have been investigated extensively, but those from brown rot fungi remain largely unknown. In this paper, a laccase isoform Pplcc2 from the brown rot fungus Postia placenta MAD-698-R was expressed heterologously in Pichia pastoris GS115, purified and the properties of the enzyme were determined. Results The molecular weight of the protein was determined to be 67 kDa using SDS-PAGE. It cannot oxidize syringaldazine (SGZ), but it can oxidize 2,2'-azino-di-(3-ethylbenzothialozin-6-Sulfonic acid) (ABTS) and 2,6-dimethoxyphenol (DMP). Specific activity for ABTS was 1960 ± 19 Unit/mg. The catalytic...
Tipo: Journal article Palavras-chave: Brown rot fungi; Pichia pastoris; Postia placenta; Thermostable laccase.
Ano: 2015 URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582015000600011
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Molecular cloning and expression of a putative crustacean hyperglycemic hormone of Litopenaeus vannamei in Pichia pastoris Electron. J. Biotechnol.
Sánchez-Castrejón,Edna; Ponce-Rivas,Elizabeth; Aguilar,Manuel B; Díaz,Fernando.
Crustacean hyperglycemic hormone (CHH) is the most abundant and best studied member of the CHH/MIH/GIH neuropeptide hormone family. CHH plays a major role in controlling glucose levels in the hemolymph, and it also has significance in regulating molting, reproduction, and osmoregulation. In contrast, molt-inhibiting hormone (MIH) is responsible for maintaining animals in an intermolt stage. In this study, Liv-MIH-1 cDNA, which encodes a mature neuropeptide from the eyestalk of white shrimp, Litopenaeus vannamei, was expressed in methylotrophic yeast (Pichia pastoris KM71) under the control of an alcohol oxidase promoter. Recombinant Liv-MIH-1 was secreted into the culture medium using the α-factor prepro-sequence without Glu-Ala repeats. The...
Tipo: Journal article Palavras-chave: CHH; Litopenaeus vannamei; Pichia pastoris; Recombinant protein.
Ano: 2008 URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582008000400009
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Performance of jundiá larvae, Rhamdia quelen, fed on probiotic supplemented diets Animal Sciences
Pinto, Vilson Borba; Costenaro-Ferreira, Cristiano; Oliveira, Paulo Leonardo Silva; Oliveira, Rodrigo Ribeiro Bezerra de; Piedras, Sérgio Renato Noguez; Pouey, Juvêncio Luis Osório Fernandes.
Since probiotics have proved to be a viable alternative to antibiotics as enhancers of animal growth, the performance, uniformity and mortality rates of the jundiá (Rhamdia quelen) larvae fed on diets with different probiotics were evaluated. Jundiá larvae, aged four days post hatching, were fed during 21 days with the following diets, in four replicates, namely, CO: control feed, without probiotics; PP: feed with Pichia pastoris; SB: feed with Saccharomyces boulardii; BT: feed with Bacillus cereus var. toyoi. Among the tested probiotic, Bacillus cereus var. toyoi had the best results due to the fact that the larvae were 25% heavier than CO at the end of the first week; the difference increased to 28% by the end of the trial. Further, BT also improved...
Tipo: Info:eu-repo/semantics/article Palavras-chave: 5.06.03.04-3 fish; Uniformity; Bacillus cereus var. toyoi; Saccharomyces boulardii; Pichia pastoris.
Ano: 2015 URL: http://periodicos.uem.br/ojs/index.php/ActaSciAnimSci/article/view/27106
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Production of L1 protein from different types of HPV in Pichia pastoris using an integrative vector BJMBR
Coimbra,E.C.; Gomes,F.B.; Campos,J.F.; D’arc,M.; Carvalho,J.C.; Mariz,F.C.; Jesus,A.L.S.; Stocco,R.C.; Beçak,W.; Freitas,A.C..
Human papillomavirus (HPV) infection is the most common sexually transmitted disease in the world and is related to the etiology of cervical cancer. The most common high-risk HPV types are 16 and 18; however, the second most prevalent type in the Midwestern region of Brazil is HPV-33. New vaccine strategies against HPV have shown that virus-like particles (VLP) of the major capsid protein (L1) induce efficient production of antibodies, which confer protection against the same viral type. The methylotrophic yeast Pichia pastoris is an efficient and inexpensive expression system for the production of high levels of heterologous proteins stably using a wild-type gene in combination with an integrative vector. It was recently demonstrated that P. pastoris can...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Human papillomavirus; Cervical cancer; Pichia pastoris; L1.
Ano: 2011 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2011001200003
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Production of leptospiral LipL32 antigen in Pichia pastoris and its use in an enzyme-linked immunosorbent assay BABT
Monte,Leonardo Garcia; Leal,Fernanda Munhoz Dos Anjos; Hartwig,Daiane Drawanz; Vasconcellos,Sílvio Arruda; Brihuega,Bibiana; Dellagostin,Odir Antonio; Hartleben,Cláudia Pinho.
The production of recombinant LipL32 protein using Escherichia coli has been used extensively for the development of vaccines and diagnostic tests for leptospirosis. However, E. coli has demonstrated limitations, including low yield and lack of post-translational modifications. In this study, rLipL32 was produced in eukaryotic expression system (Pichia pastoris) and evaluated the antigen by enzyme-linked immunosorbent assay (ELISA). The yield obtained from the culture supernatant reached 270 mg/L and ELISA showed an accuracy of 95.34%. In summary, the production of rLipL32 using P. pastoris did not impair the antigenic characteristics of this antigen and ensured its use for detecting the leptospiral antibodies in swine sera.
Tipo: Info:eu-repo/semantics/article Palavras-chave: Pichia pastoris; LipL32; ELISA; Leptospirosis.
Ano: 2014 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132014000300007
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Production of recombinant human epidermal growth factor in Pichia pastoris BJM
Eissazadeh,Samira; Moeini,Hassan; Dezfouli,Maryam Ghandizadeh; Heidary,Somayyeh; Nelofer,Rubina; Abdullah,Mohd Puad.
Abstract This study was carried out to express human epidermal growth factor (hEGF) in Pichia pastoris GS115. For this aim, the hEGF gene was cloned into the pPIC9K expression vector, and then integrated into P. pastoris by electroporation. ELISA-based assay showed that the amount of hEGF secreted into the medium can be affected by the fermentation conditions especially by culture medium, pH and temperature. The best medium for the optimal hEGF production was BMMY buffered at a pH range of 6.0 and 7.0. The highest amount of hEGF with an average yield of 2.27 µg/mL was obtained through an induction of the culture with 0.5% (v/v) methanol for 60 h. The artificial neural network (ANN) analysis revealed that changes in both pH and temperature significantly...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Pichia pastoris; Human epidermal growth factor; Artificial neural network.
Ano: 2017 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822017000200286
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