| Full text | |
| Author(s): |
Gabas, Mariana L.
;
Otvos, Luca P.
;
Almeida, Naira B. O.
;
Farias, Sofia O.
;
Val, Giovanna S.
;
Machado, Luciana E. S. F.
Total Authors: 6
|
| Document type: | Journal article |
| Source: | Biochemical and Biophysical Research Communications; v. 799, p. 9-pg., 2026-02-05. |
| Abstract | |
Molecular cloning and heterologous protein expression are essential for investigating protein function and interactions with ligands such as small molecules, drugs, and other proteins. Studies on the redox regulation, intermolecular interactions, structural determination, and structural dynamics of mitochondrial protein phosphatases require high-yield expression of soluble, catalytically active enzymes. Accordingly, the aim of this study was to optimize the cloning, expression, and soluble purification of mitochondrial protein phosphatases in their monomeric and active forms. We designed 22 expression constructs encoding the mitochondrial protein phosphatases PTPMT1, PP2Cm, PPTc7, and PGAM5, incorporating variations with or without the mitochondrial targeting sequence (MTS) and solubility-enhancing fusion tags. Our results demonstrate that, for matrix localized phosphatases, MTS removal combined with a soluble fusion tag is essential for obtaining soluble, structurally stable, properly folded, and catalytically active proteins. In contrast, intermembrane space phosphatase PGAM5 was well structured and active across constructs, thoug MTS presence reduced expression yields and increased protein instability. Overall, this work underscores the critical role of rational construct design for the successful production of mitochondrial protein phosphatases suitable for in vitro biochemical and structural studies. (AU) | |
| FAPESP's process: | 21/13213-9 - Redox regulation of mitochondrial protein phosphatases manganese-dependent and investigation of their network interactions |
| Grantee: | Naira Bellissimo Oliveira de Almeida |
| Support Opportunities: | Scholarships in Brazil - Scientific Initiation |
| FAPESP's process: | 22/02313-5 - Analysis of the ligands and the interaction network of the mitochondrial protein phosphatase PGAM5 |
| Grantee: | Sofia de Oliveira Farias |
| Support Opportunities: | Scholarships in Brazil - Scientific Initiation |
| FAPESP's process: | 21/10474-6 - Identification of mitochondrial DUSPs ligands through bioinformatics tools |
| Grantee: | Luca Paulino Otvos |
| Support Opportunities: | Scholarships in Brazil - Scientific Initiation |
| FAPESP's process: | 20/10168-0 - Structural and functional characterization of mitochondrial protein phosphatases by redox metabolism and their role in cell biology |
| Grantee: | Luciana Elena de Souza Fraga Machado |
| Support Opportunities: | Scholarships in Brazil - Young Researchers |
| FAPESP's process: | 22/04064-2 - Mitochondrial protein phosphatases cloning, expression and purification |
| Grantee: | Mariana Louise Gabas |
| Support Opportunities: | Scholarships in Brazil - Technical Training Program - Technical Training |
| FAPESP's process: | 23/02968-4 - Redox and structural regulation of Protein Tyrosine Phosphatase Mitochondrial 1 (PTPMT1) |
| Grantee: | Luca Paulino Otvos |
| Support Opportunities: | Scholarships in Brazil - Master |
| FAPESP's process: | 19/02605-3 - Structural and functional characterization of mitochondrial protein phosphatases by redox metabolism and their role in cell biology |
| Grantee: | Luciana Elena de Souza Fraga Machado |
| Support Opportunities: | Research Grants - Young Investigators Grants |