Expression of the human carboxylesterase 2 enzyme (CES2) in mammalian cells

• Author(s): Cunha, Bárbara Martins Paiva da
• Date: 2011
• Persistent ID: http://hdl.handle.net/10362/6659
• Origin: Repositório Institucional da UNL
• Subject(s): Carboxylesterase 2; Manufacturing; Secretion; Mammalian cells; Scale-up
• Document Type: Master thesis
• Language: English
• Advisor(s): Simplício, Ana; Coroadinha, Ana
• Contributor(s): RUN

Description

Dissertation to obtain a Master Degree in Biotechnology

The carboxylesterase 2 enzyme (CES2), the main carboxylesterase (CES) expressed in human intestine, has a key role in the metabolism of ester containing xenobiotics including the activation of several prodrugs and therefore, it can influence significantly the bioavailability of these compounds. CES2 is receiving an increasing attention due to its potential application in anti-cancer combined therapies, for the treatment of different pathologies like colon adenocarcinoma and malignant glioma. Unlike carboxylesterase 1 (CES1), the structure of this enzyme is not yet known. Since the knowledge of the structure of this protein will be crucial for the understanding of its properties, it becomes relevant the establishment of a process for the production and purification at the milligram level. The development and optimization of human recombinant CES2 production process, using human embryonic kidney cells (HEK-293T cells), is described in this thesis. First, the C-terminal 10xHistidine tag CES2 (CES2-10xHis) was produced using adherent cultures, and then the process was transferred to suspension cultures with serum-free media and finally scaled up to a five-litre stirred tank bioreactor. An affinity chromatography purification process was applied to obtain the CES2-10xHis protein, with a high purity grade. Despite being an Endoplasmic Reticulum (ER) anchored protein, secretion of CES2 to serum free media was achieved due to the presence of the in frame C-terminal 10xHistidine tag. With this elegant procedure we avoided the addition of extra N-terminal signaling sequences or the mutation or deletion of the C-terminal Histidine - Threonine - Glutamic acid - Leucine (HTEL) motif responsible for anchoring the protein in the lumen of the ER. Secretion contributed to a good production yield; however, the global process yield was quite low, due to difficulties in the purification step, which we attributed to protein aggregation.