Chagasin from Trypanosoma cruzi as a molecular scaffold to express epitopes of TSA-1 as soluble recombinant chimeras.

Autor: Cárdenas-Guerra RE; Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Av. IPN # 2508, Col. San Pedro Zacatenco, Gustavo A. Madero, CP 07360, Mexico City, Mexico., Montes-Flores O; Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Av. IPN # 2508, Col. San Pedro Zacatenco, Gustavo A. Madero, CP 07360, Mexico City, Mexico., Nava-Pintor EE; Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Av. IPN # 2508, Col. San Pedro Zacatenco, Gustavo A. Madero, CP 07360, Mexico City, Mexico., Reséndiz-Cardiel G; Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Av. IPN # 2508, Col. San Pedro Zacatenco, Gustavo A. Madero, CP 07360, Mexico City, Mexico., Flores-Pucheta CI; Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Av. IPN # 2508, Col. San Pedro Zacatenco, Gustavo A. Madero, CP 07360, Mexico City, Mexico., Rodríguez-Gavaldón YI; Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Av. IPN # 2508, Col. San Pedro Zacatenco, Gustavo A. Madero, CP 07360, Mexico City, Mexico., Arroyo R; Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Av. IPN # 2508, Col. San Pedro Zacatenco, Gustavo A. Madero, CP 07360, Mexico City, Mexico., Bottazzi ME; Texas Children's Hospital Center for Vaccine Development, Department of Pediatrics and Molecular Virology and Microbiology, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX, USA., Hotez PJ; Texas Children's Hospital Center for Vaccine Development, Department of Pediatrics and Molecular Virology and Microbiology, National School of Tropical Medicine, Baylor College of Medicine, Houston, TX, USA., Ortega-López J; Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Av. IPN # 2508, Col. San Pedro Zacatenco, Gustavo A. Madero, CP 07360, Mexico City, Mexico. Electronic address: jortega@cinvestav.mx.
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2024 Jun; Vol. 218, pp. 106458. Date of Electronic Publication: 2024 Feb 27.
DOI: 10.1016/j.pep.2024.106458
Abstrakt: Trypanosoma cruzi is the causative agent of Chagas disease, a global public health problem. New therapeutic drugs and biologics are needed. The TSA-1 recombinant protein of T. cruzi is one such promising antigen for developing a therapeutic vaccine. However, it is overexpressed in E. coli as inclusion bodies, requiring an additional refolding step. As an alternative, in this study, we propose the endogenous cysteine protease inhibitor chagasin as a molecular scaffold to generate chimeric proteins. These proteins will contain combinations of two of the five conserved epitopes (E1 to E5) of TSA-1 in the L4 and L6 chagasin loops. Twenty chimeras (Q1-Q20) were designed, and their solubility was predicted using bioinformatics tools. Nine chimeras with different degrees of solubility were selected and expressed in E. coli BL21 (DE3). Western blot assays with anti-6x-His and anti-chagasin antibodies confirmed the expression of soluble recombinant chimeras. Both theoretically and experimentally, the Q12 (E5-E3) chimera was the most soluble, and the Q20 (E4-E5) the most insoluble protein. Q4 (E5-E1) and Q8 (E5-E2) chimeras were classified as proteins with medium solubility that exhibited the highest yield in the soluble fraction. Notably, Q4 has a yield of 239 mg/L, well above the yield of recombinant chagasin (16.5 mg/L) expressed in a soluble form. The expression of the Q4 chimera was scaled up to a 7 L fermenter obtaining a yield of 490 mg/L. These data show that chagasin can serve as a molecular scaffold for the expression of TSA-1 epitopes in the form of soluble chimeras.
Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest.
(Copyright © 2024 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
Externí odkaz: