Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase


Catechol-O-methyltransferase (COMT) has been involved in a number of medical conditions including catechol-estrogen-induced cancers and a great range of cardiovascular and neurodegenerative diseases such as Parkinson's disease. Currently, Parkinson's disease treatment relies on a triple prophylaxis, involving dopamine replacement by levodopa, the use of aromatic L-amino acid decarboxylase inhibitors, and the use of COMT inhibitors. Typically, COMT is highly thermolabile, and its soluble isoform (SCOMT) loses biological activity within a short time span preventing further structural and functional trials. Herein, we characterized the thermal stability profile of lysate cells from Komagataella pastoris containing human recombinant SCOMT (hSCOMT) and enzyme-purified fractions (by Immobilized Metal Affinity Chromatography-IMAC) upon interaction with several buffers and additives by Thermal Shift Assay (TSA) and a biological activity assessment. Based on the obtained results, potential conditions able to increase the thermal stability of hSCOMT have been found through the analysis of melting temperature (T-m) variations. Moreover, the use of the ionic liquid 1-butyl-3-methylimidazolium chloride [C(4)mim]Cl (along with cysteine, trehalose, and glycerol) ensures complete protein solubilization as well as an increment in the protein Tm of approximately 10 degrees C. Thus, the developed formulation enhances hSCOMT stability with an increment in the percentage of activity recovery of 200% and 70% when the protein was stored at 4 degrees C and -80 degrees C, respectively, for 12 h. The formation of metanephrine over time confirmed that the enzyme showed twice the productivity in the presence of the additive. These outstanding achievements might pave the way for the development of future hSCOMT structural and biophysical studies, which are fundamental for the design of novel therapeutic molecules.




Biochemistry & Molecular Biology; Chemistry


Goncalves, AM; Pedro, AQ; Oliveira, DM; Oliveira, AE; Santos, MFA; Correia, MAS; Queiroz, JA; Gallardo, E; Romao, MJ; Passarinha, LA

nossos autores


This work was supported by Laboratorio de Farmaco-Toxicologia, UBIMedical, and by Centro de Competencias em Cloud Computing, co-financed by the European Regional Development Fund (ERDF) through the Programa Operacional Regional do Centro (Centro 2020), in the scope of the Sistema de Apoio a Investigacao Cientifica e Tecnologica-Programas Integrados de IC&DT (Centro-01-0145-FEDER-000019-C4). This work was developed within the scope of the CICS-UBI (projects UIDB/00709/2020, UIDP/00709/2020), CICECO-Aveiro Institute of Materials (projects UIDB/50011/2020, UIDP/50011/2020 & LA/P/0006/2020), the Applied Molecular Biosciences Unit UCIBIO (UIDB/04378/2020 and UIDP/04378/2020) and the Associate Laboratory Institute for Health and Bioeconomy-i4HB (project LA/P/0140/2020), which are financed by National Funds from FCT/MCTES. Researchers also acknowledge funding by FEDER through COMPETE 2020-Programa Operacional Competitividade e Internacionalizacao (POCI), and by national funds (OE), through FCT/MCTES from the project IL2BioPro-PTDC/BII-BBF/30840/2017. Ana M. Goncalves's individual PhD Fellowship (SFRH/BD/147519/2019), Augusto Q. Pedro research contract CEEC-IND/02599/2020 under the Scientific Stimulus-Individual Call, and Luis A. Passarinha's sabbatical fellowship (SFRH/BSAB/150376/2019) from the Portuguese Foundation for Science and Technology (FCT) within the scope of POCH-Advanced Formation programs co-funded by European Social Fund and MCTES.

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