Navegando por Palavras-chave "Pichia pastoris"
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- ItemSomente MetadadadosCloning and expression of a functionally active truncated N-glycosylated KSHVORF4/KCP/Kaposica in the methylotrophic yeast Pichia pastoris(New York Acad Sciences, 2005-01-01) Pereira, NAG; Juliano, M. A.; Carmona, A. K.; Sturrock, E. D.; Kotwal, G. J.; Lahiri, D. K.; Univ Cape Town; Universidade Federal de São Paulo (UNIFESP)Kaposi's sarcoma herpesvirus (KSHV) is a typical DNA virus that is associated with a number of proliferative diseases including Kaposi's sarcoma. the KSHV open reading frame (ORF) 4 encodes a complement regulatory protein (Kaposi complement control protein, KCP) that binds complement components and inhibits the complement-mediated lysis of cells infected by the virus, thus providing a strategy for evasion of the host complement system. Based on primary sequence analysis and comparison with other functionally and structurally similar proteins, oligonucleotide primers were designed to amplify by polymerase chain reaction (PCR) three regions of the predicted ORF 4 from human herpes virus-8 (HHV-8) DNA isolated from a primary effusion lymphoma cell line. the PCR products were inserted by ligation into the expression vector pPIC9 to generate three recombinant plasmids for heterologous expression in the yeast, Pichia pastoris, to produce separately the four N-terminal sushi domains (KCP-S, small), KCP protein lacking the putative transmembrane-binding domain (KCP-M, medium), and the full-length protein (KCP-F, full). Expression of the viral proteins was confirmed by SDSPAGE, immunologic detection, and Western blot analyses using a rabbit polyclonal antibody directed against a selected peptide region that is common to all three recombinant KCPs. KCP-S directly from expression media could inhibit complement-mediated lysis of sensitized sheep erythrocytes by approximately 60% in a hemolysis assay. This result confirms previous reports that recombinant KCP is twice as efficient in inhibiting the classic pathway-mediated lysis of erythrocytes than is the vaccinia virus complement control protein, which also contains four sushi domains.
- ItemRestritoHigh expression of human carboxypeptidase M in Pichia pastoris. Purification and partial characterization(Assoc Bras Divulg Cientifica, 2006-02-01) Craveiro, Rogerio Bastos [UNIFESP]; Ramalho, João Daivison Silva [UNIFESP]; Chagas, Jair Ribeiro [UNIFESP]; Wang, Pamella Huey Mei [UNIFESP]; Casarini, Dulce Elena [UNIFESP]; Pesquero, Jorge Luiz [UNIFESP]; Araujo, Ronaldo Carvalho [UNIFESP]; Pesquero, João Bosco [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Mogi Cruzes; Universidade Federal de Minas Gerais (UFMG)Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidylinositol-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 cloning and transfection, was integrated into the yeast genome, which produced the active protein. the recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. the enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein(-1) min(-1)). This purification protocol permitted us to obtain 410 mg of the purified protein per titer of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes.
- ItemAcesso aberto (Open Access)Produção e Caracterização in silico da Proteína Inibidora de elastase de neutrófilos (NEI) recombinante do carrapato Rhipicephalus microplus: uma candidata para tratamento de desordens inflamatórias(Universidade Federal de São Paulo, 2021-06-02) Leite, Gabriela Karen [UNIFESP]; Campos, Claudia Barbosa Ladeira de [UNIFESP]; Anatriello, Elen; http://lattes.cnpq.br/6295907986341326; http://lattes.cnpq.br/8867737169235839; http://lattes.cnpq.br/4772381910969440Os carrapatos, tais quais o Rhipicephalus microplus, são artrópodes ectoparasitas hematófagos, que precisam permanecer fixos aos seus hospedeiros durante todo o tempo de vida para se alimentarem. Para tal, secretam diversas moléculas em sua saliva a fim de evadir a resposta imune do hospedeiro. Ao estudar essas diferentes moléculas, observa-se uma grande gama de potenciais imunobiológicos que podem atuar como efetores farmacológicos contra desordens inflamatórias causadas ou não por patógenos. Um dos desafios está em estabelecer formas de produção destas moléculas com baixo custo e grande rendimento. O objetivo do trabalho foi a produção da proteína inibidora de elastase de neutrófilo (NEI) recombinante em Escherichia coli e em Pichia pastoris, cuja sequência primária foi derivada do sialotranscriptoma do carrapato R. microplus, e a sua caracterização in silico. Conduzimos esse projeto, comparando os dois sistemas de produção de proteínas recombinantes, em pequena e larga escala em laboratório. Também foram realizados métodos de purificação como a cromatografia por afinidade e por gel filtração, para posteriormente ser utilizada na investigação do seu potencial farmacológico em modelos experimentais murinos. Para validar esse potencial, comparamos a nossa proteína de interesse com proteínas já caracterizadas como Inibidoras de elastase de carrapatos como R. microplus, A. monolakensis, I. scapularis, H. asiaticum, I. persulcatus, O. parkeri, H. dromedarii H. marginatum rufipe, A. delacruzi, A. triste, e A. cajennense e A. parvvum assim como do mosquito C. tarsalis. Esse comparativo foi feito via comparações filogenéticas e estruturais em vários níveis (verificando estruturas primarias, secundarias e tridimensionais). Nesse projeto, revistando os principais processos biotecnológicos de produção de proteínas recombinantes usando técnicas como crescimento em meio LB, usando a via do OperonLac e para concentração de proteínas por diálise usado no processo de produção via E. coli competentes, assim como crescendo em meios específicos com protocolos de produção proteica otimizados, ativando a produção da proteína de interesse usando metanol e usando instrumentos como NanoDrop para garantir a concentração a proteína produzida conseguiu-se definir o protocolo de P. pastoris como o de melhor rendimento e fácil purificação para utilização em modelos in vivo. Os resultados deste trabalho podem contribuir com um potencial candidata para tratamento de doenças inflamatórias com exacerbação da ação neutrofílica.
- ItemSomente MetadadadosRecombinant expression and characterization of a cysteine peptidase from Xanthomonas citri subsp citri(Funpec-editora, 2012-01-01) Soares-Costa, Andrea; Silveira, Roseli Santos da; Novo, Maria Teresa Marques; Alves, Marcio Fernando Madureira [UNIFESP]; Carmona, Adriana Karaoglanovic [UNIFESP]; Belasque Junior, José; Henrique-Silva, Flávio; Universidade Federal de São Carlos (UFSCar); Universidade Federal de São Paulo (UNIFESP); Fundo Def CitriculturaXanthomonas citri subsp citri (Xac) is the bacterium responsible for citrus canker disease in citrus plants. the aim of this study was to describe the recombinant expression, purification, and characterization of a cysteine peptidase from Xac strain 306, which is a candidate for involvement in the pathogenicity of this bacterium. the gene was cloned and expressed in Pichia pastoris, and the cysteine peptidase was successfully expressed, secreted, and purified using affinity chromatography with a yield of approximately 10 mg/L. A polyclonal antibody produced against cysteine peptidase from X. citri subsp citri fused with HIS tag ((HIS)CPXAC) recognized the purified recombinant cysteine peptidase (HIS)CPXAC, confirming the correct production of this protein in P. pastoris. the same antibody detected the protein in the culture supernatant of Xac grown in pathogenicity-inducing medium. Kinetic analysis revealed that (HIS)CPXAC hydrolyzed the carbobenzoxy-Leu-Arg-7-amido-4-methylcoumarin substrate with a catalytic efficiency (k(cat)/K-m) of 47 mu M-1.s(-1). the purified (HIS)CPXAC displayed maximal catalytic activity at pH 5.5 and 30 degrees C. the recombinant enzyme was inhibited by the specific cysteine peptidase inhibitor E-64, as well as by the recombinant cysteine peptidase inhibitors CaneCPI-1, CaneCPI-2, CaneCPI-3, and CaneCPI-4, with K-i values of 1.214, 84.64, 0.09, 0.09, and 0.012 nM, respectively. Finally, the N-terminal sequencing of the purified protein enabled the identification of the first 5 amino acid residues (AVHGM) immediately after the putative signal peptide, thereby enabling the identification of the cleavage point and corroborating previous studies that have identified this sequence in a secreted protein from Xanthomonas spp.