TY - JOUR
T1 - Thermofluor-Based Optimization Strategy for the Stabilization of Recombinant Human Soluble Catechol-O-Methyltransferase
AU - Gonçalves, Ana M.
AU - Pedro, Augusto Q.
AU - Oliveira, Diana M.
AU - Oliveira, Adriana E.
AU - Santos, Marino F. A.
AU - Correia, Márcia A. S.
AU - Queiroz, João A.
AU - Gallardo, Eugénia
AU - Romão, Maria J.
AU - Passarinha, Luís A.
N1 - info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F00709%2F2020/PT#
info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDP%2F00709%2F2020/PT#
info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F50011%2F2020/PT#
info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDP%2F50011%2F2020/PT#
info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F04378%2F2020/PT#
info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDP%2F04378%2F2020/PT#
info:eu-repo/grantAgreement/FCT/9471 - RIDTI/PTDC%2FBII-BBF%2F30840%2F2017/PT#
info:eu-repo/grantAgreement/FCT/OE/SFRH%2FBD%2F147519%2F2019/PT#
info:eu-repo/grantAgreement/FCT/OE/SFRH%2FBSAB%2F150376%2F2019/PT#
Funding Information:
This work was supported by Laboratório de Fármaco-Toxicologia, UBIMedical, and by Centro de Competências 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 à Investigação Científica e Tecnológica-Programas Integrados de IC&DT (Centro-01-0145-FEDER-000019-C4). This work was developed within the scope of the CICECO-Aveiro Institute of Materials (projects LA/P/0006/2020 ), and the Associate Laboratory Institute for Health and Bioeconomy—i4HB (project LA/P/0140/2020), which are financed by National Funds from FCT/MCTES. Augusto Q. Pedro research contract CEEC-IND/02599/2020 under the Scientific Stimulus—Individual Call 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.
Publisher Copyright:
© 2022 by the authors.
PY - 2022/10
Y1 - 2022/10
N2 - 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 (Tm) variations. Moreover, the use of the ionic liquid 1-butyl-3-methylimidazolium chloride [C4mim]Cl (along with cysteine, trehalose, and glycerol) ensures complete protein solubilization as well as an increment in the protein Tm of approximately 10 °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 °C and −80 °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.
AB - 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 (Tm) variations. Moreover, the use of the ionic liquid 1-butyl-3-methylimidazolium chloride [C4mim]Cl (along with cysteine, trehalose, and glycerol) ensures complete protein solubilization as well as an increment in the protein Tm of approximately 10 °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 °C and −80 °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.
KW - protein activity
KW - protein stability
KW - soluble catechol-O-methyltransferase (SCOMT)
KW - thermal shift assay (TSA)
UR - http://www.scopus.com/inward/record.url?scp=85140810103&partnerID=8YFLogxK
U2 - 10.3390/ijms232012298
DO - 10.3390/ijms232012298
M3 - Article
C2 - 36293152
AN - SCOPUS:85140810103
SN - 1661-6596
VL - 23
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
IS - 20
M1 - 12298
ER -