Development of a new recombinant protein to protect against tooth demineralization and evaluation of the expression system in Pichia pastoris yeast

• Grant number: 24/20780-5
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: May 01, 2025
• End date: April 30, 2026
• Field of knowledge: Health Sciences - Dentistry - Social and Preventive Dentistry
• Principal Investigator: Marília Afonso Rabelo Buzalaf
• Grantee: Valentine Spagnol
• Host Institution: Centro de Ciências Biológicas e da Saúde (CCBS). Universidade Federal de São Carlos (UFSCAR). São Carlos , SP, Brazil
• Associated research grant: 19/26070-1 - Modulation of acquired enamel pellicle to control dental mineral loss: unravelling mechanisms to make therapies possible, AP.TEM

Abstract

Efficient production of recombinant proteins is essential to meet the growing demands for research and clinical application. Currently, three proteins investigated by our group, CaneCPI-5, MaquiCPI-3, and CaneStat, with potential for incorporation into the acquired enamel pellicle (AEP) for protection against erosive dental wear and caries, are expressed in the Escherichia coli bacterial system. Although widely used, this system has some limitations, especially when seeking scalability and purity, requiring expensive purification steps. In this project, we propose the transition to the Pichia pastoris yeast system, capable of reaching high cell densities and secreting the recombinant protein directly into the culture medium, in addition to testing whether, due to this advantage of the system, it will be possible to obtain the proteins from the total supernatant dialyzed by tangential flow filtration (TFF) and spray dried, in order to eliminate the chromatography purification step and reduce process costs. In parallel, we also propose the construction of a new molecule, a concatamer formed by seven units of the statherin peptide, aiming to enhance its capacity for adhesion and protection to enamel, and to express it in the same system. The process will be conducted on a bench scale, using shakers, and the proteins produced will be validated for their capacity to protect against initial erosion in vitro. Additionally, the capacity of the proteins to incorporate into the PAE and protect enamel against erosion in situ will be evaluated during an internship abroad (BEPE grant, to be requested in due course). At the end of this project, we expect to enable a new expression system for the proteins studied by our group, capable of meeting the expected increases in their demand, given the advances in the phase of clinical research. We also expect that the new proposed protein, the concatamer of the statherin peptide, will present a greater capacity for binding to PAE compared to the protective effect already observed with the isolated peptide.