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Master's Dissertation
DOI
Document
Author
Full name
Henrique Pellin Biasoto
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2019
Supervisor
Committee
Monteiro, Gisele (President)
Oliveira, Marcos Antonio de
Pessoa Junior, Adalberto
Soares, Irene da Silva
Title in Portuguese
Expressão da L-asparaginase II de Saccharomyces cerevisiae recombinante extracelular com glicosilação humanizada em Pichia pastoris
Keywords in Portuguese
Asparaginase
Expressão extracelular
Leucemia linfoide aguda
Pichia pastoris
Proteína recombinante
Purificação enzimática
Saccharomyces cerevisiae
Abstract in Portuguese
L-asparaginase é um inibidor eficiente do crescimento tumoral, usado em sessões de quimioterapia contra a Leucemia Linfoblástica Aguda (LLA), resultando na remissão completa da doença em 90% dos pacientes tratados. A L-asparaginase II de Saccharomyces cerevisiae (ScASNaseII) tem alto potencial de superar os efeitos adversos da L-asparaginase de bactéria, porém sua produção endógena resulta em uma proteína hipermanosilada e, consequentemente, imunogênica. A cepa de Pichia pastoris Glycoswitch tem a maquinaria para expressar e secretar altas quantidades de enzima com glicosilação humanizada. Nesse trabalho, descrevemos o processo genético para expressar a ScASNaseII no meio extracelular pela P. pastoris Glycoswitch, e também os parâmetros bioquímicos, perfil cinético, citotoxicidade contra células leucêmicas e a interferência da glicosilação na atividade da enzima obtida. Nossos dados mostram que a cepa aplicada foi capaz de expressar ScASNaseII no meio extracelular passível de purificação de proteínas contaminantes com apenas um passo cromatográfico. A atividade específica para asparagina foi 218,2 UI/mg e a atividade glutaminásica representou 3,1% da atividade asparaginásica. Os parâmetros cinéticos foram KM = 120,5 µM e a eficiência catalítica de 3,8 x 105 M-1s-1. Análises por meio de gel nativo sugerem uma conformação tetramérica de aproximadamente 150 kDa. Essa é uma nova estratégia de produzir essa enzima de forma extracelular, com mais facilidade de purificação e com melhores propriedades biotecnológicas.
Title in English
Extracellular expression of Saccharomyces cerevisiae's L-asparaginase II in Pichia pastoris with humanized glycosylation
Keywords in English
Acute lymphoid leukemia
Asparaginase
Enzyme purification
Extracellular expression
Pichia pastoris
Recombinant protein
Saccharomyces cerevisiae
Abstract in English
L-asparaginase is an efficient inhibitor of tumor development, used in chemotherapy sessions against acute lymphoblastic leukemia (ALL) tumor cell; its use results in 90% complete remission of the disease in treated patients. Saccharomyces cerevisiae's L-asparaginase II (ScASNaseII) has a high potential to overcome the side effects of bacteria L-asparaginase, but the endogenous production of it results in hypermannosylated immunogenic enzyme. However, Pichia pastoris Glycoswitch strain has the machinery to express and secrete high quantity of the enzyme and with humanized glycosylation. Here we describe the genetic process to acquire the ScASNaseII in the extracellular medium expressed by P. pastoris Glycoswitch, and the biochemical properties of the resultant enzyme, kinetic profile, cytotoxicity against ALL cell line and the interference of glycosylation in its activity. Our data show that the strain employed is able to express extracellular asparaginase active and possible to be purified of contaminant proteins using a single chromatographic step. The specific activity using asparagine was 218.2 IU.mg-1 and the glutaminase activity represents 3.1% of its asparaginase activity. The kinetics parameters were KM=120.5 µM and a catalytic efficiency of 3.8x105 M-1s-1. The Native-PAGE suggested a tetrameric protein conformation, with approximately 150 kDa. This is a novel strategy to produce this enzyme extracellularly, easier to purify and with better biotechnological properties.
 
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Publishing Date
2019-10-18
 
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