Carboxylesterase 2 production and characterization in human cells: new insights into enzyme oligomerization and activity
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Carboxylesterase 2 (CES2), the main carboxylesterase expressed in human intestine, is an increasingly important enzyme in anti-cancer combined therapies for the treatment of different pathologies like colon adenocarcinoma and malignant glioma. The production of human recombinant CES2, in human embryonic kidney cells (HEK-293T cells) using serum-free media, is herein described. CES2 secretion to the media was achieved by the simple addition of an in-frame C-terminal 10× histidine tag (CES2-10xHis) without the need of addition of extra N-terminal signalling sequences or the mutation or deletion of the C-terminal HTEL motif responsible for retaining the protein in the lumen of endoplasmic reticulum. This secretion allowed a fourfold increase in CES2 production. The characterization of human recombinant CES2 showed that this protein exists in other active and inactive forms than the described 60 kDa monomer.
KeywordsCarboxylesterases Human cells Protein expression Enzyme characterization
This work was funded by Fundação para a Ciência e Tecnologia, Portugal (SFRH/BD/44025/2008, PTDC/EBB-BIO/111530/2009, PEst-OE/EQB/LA0004/2011). The authors wish to thank Dr. C. Frazão, Dr. T. M. Bandeiras and Dr. P. M. Matias for the fruitful discussions, and Dr. Júlia Costa from ITQB, Oeiras for the expertise in glycosylation.
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