Clonagem e expressão da glucocerebrosidase humana em células de ovário de hamster chinês (CHO). / Cloning and expression of human glucocerebrosidase in Chinese hamster ovary (CHO) cells.

AUTOR(ES)
DATA DE PUBLICAÇÃO

2010

RESUMO

Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gauchers disease. Current treatment consists on enzyme replacement therapy by the administration of recombinant GCR produced in CHO cells. However, the medicine available in the market is extremely expensive. In this work, we proposed the cloning and expression of human GCR in CHO cells, in order to obtain a productive cellular clone for future production of GCR enzyme at a lower cost at the Butantan Institute. The stable expression of recombinant GCR was obtained after transfection of CHO-dhfr- cells with pED mammalian expression vector containing the GCR cDNA, followed by gene amplification with MTX. The GCR was detected by western blotting analysis, either as cell-associated (~ 64 kDa) or as secreted forms (63-69 kDa), using the anti-GCR polyclonal antibody produced in this work. The secreted enzyme was active on 4-MUG and was produced at a level of about 5,14 pg/cell/day for the best producer subclone, selected for subsequent steps of GCR production on large scale in next future.

ASSUNTO(S)

glicoproteínas lysosomes dhfr expressão dicistrônica glycopeptides glycoproteins amplificação de genes glicopeptídeos lisossomos ovário dhfr cho cells glucocerebrosidase recombinant proteins dicistronic expression expressão gênica gauchers disease glucocerebrosidase células cho genes amplification gene expression doença de gaucher ovary proteínas recombinantes

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