BR-102024017701-A2 - Production process of recombinant protein from the SARS-CoV-2 RBD domain fused to the hydrophobin tail using a plant platform, the protein obtained and its use.

BR102024017701A2BR 102024017701 A2BR102024017701 A2BR 102024017701A2BR-102024017701-A2

Abstract

This invention relates to the efficient production of recombinant SARS-CoV-2 RBD domain protein fused to a hydrophobin tail on a plant platform. The process involves infiltrating Nicotiana benthamiana leaves with Agrobacterium tumefaciens containing the plasmid of interest, followed by standardized leaf collection, maceration with liquid nitrogen, semi-purification with Triton X-114, and purity assessment by electrophoresis and Western blotting. Furthermore, the functionality of the RBD-HFBI was verified by ELISA. The hydrophobin fusion allows for increased yield, stability, and ease of protein purification. In addition, the methodology utilizes an Aqueous Biphasic System (ATPS) for purification, replacing more expensive methods. The produced protein has been shown to be recognized by antibodies, making it suitable for diagnosis, therapies, and vaccines related to SARS-CoV-2. This invention represents a significant advance in the economical production of recombinant antigens and contributes to the research and development of innovative solutions in the fight against viral diseases.

Inventors

DANIEL FREIRE LIMA
MARIA IZABEL FLORINDO GUEDES
AMAURI BARBOSA DA SILVA JUNIOR
BRUNO BEZERRA DA SILVA
REINALDO BARRETO ORIÁ
ARNALDO SOLHEIRO BEZERRA
MAURICIO FRAGA VAN TILBURG
CÍCERO MATHEUS LIMA AMARAL
ROBERIO GOMES OLINDA

Assignees

FUNDAÇÃO UNIVERSIDADE ESTADUAL DO CEARÁ - FUNECE
UNIVERSIDADE FEDERAL DO CEARA

Dates

Publication Date: 20260310
Application Date: 20240828

Claims (10)

1. PRODUCTION OF RECOMBINANT SARS-CoV-2 RBD DOMAIN PROTEIN FUSIONED TO A HYDROPHOBIN TAIL USING A PLANT-BASED PLATFORM, characterized by a process comprising the following steps: a) cloning of the SARS-CoV-2 RBD domain gene sequence into a specific expression vector; b) transformation of expression plasmids into Agrobacterium tumefaciens; c) inoculation process in the plant, collection and purification of the RBD-HFBI protein using an Aqueous Biphasic System.
2. Protein according to claim 1, characterized by being fused to different tags, targeting various expression sites in compartments of eukaryotic systems.
3. Protein according to claim 1, characterized by being fused or not to hydrophobins and/or elastins or other purification tags or solubility tags.
4. Process for producing recombinant protein as defined in claim 1, characterized by comprising purification by non-chromatographic methods or other methods.
5. Process according to claim 4, characterized by being by techniques applied to HFBI or ELP tags or any other purification method.
6. Process according to claim 5, characterized in that the synthetic gene is cloned to a eukaryotic expression plasmid.
7. Process according to claim 5, characterized in that the expression system is eukaryotic.
8. Process according to claim 5, characterized in that it is carried out in plants, cell culture, fungi or any other eukaryotic organism.
9. Use of the protein as defined in claim 1, characterized by being in the preparation of an immunogen against SARS-CoV-2 and other Coronaviruses in different species, preferably humans and canines.
10. Use of recombinant protein RBD-HFBI, as described in claim 2, characterized by being used as a biotechnological input in the production of diagnostic tests for coronavirus in humans and other animals and vaccines for SARS-CoV-2.

Description

FIELD OF THE INVENTION [01] This new method relates to the area of biotechnology and biomedical research. It is made from recombinant DNA technology, using a plant platform as a bioreactor. This innovation has potential applications in the development of vaccines and diagnostic devices related to the SARS-CoV-2 virus. BACKGROUND OF THE INVENTION [02] Currently, there are several approaches to the production of recombinant proteins, including those related to the RBD domain of the SARS-CoV-2 virus. Several methods have been used, such as expression in bacterial cells, mammalian cells, or yeast expression systems. In addition, the use of plants as a platform for the production of recombinant proteins has also been explored, with Nicotiana benthamiana L. being one of the most commonly used plant species in this context. [03] One of the common strategies is the use of expression vectors based on the 35S promoter of cauliflower mosaic virus (CaMV) combined with NOS transcription terminators. Purification systems, such as column chromatography, are also used to obtain recombinant proteins in high purity (Sci., 1990, v. 250, n. 4983; Sci., 1987, v. 236, n. 4806). [04] However, these traditional methods face significant challenges regarding the efficiency of production and purification of recombinant proteins. Purification can be particularly difficult and expensive, both in terms of financial cost and time, limiting the applicability of these approaches on a large scale. In addition, the need for complex expression systems, such as animal or bacterial cells, can increase the costs and complexity of production (Front. Plant. Sci., 2020, v. 10, n. 1674; Biotec. Adv., 2020, v. 29, n. 2; BMC Biotec., 2014, v. 14, n. 1). [05] The solution proposed by the present invention is the production of the recombinant protein of the SARS-CoV-2 RBD domain using the plant platform Nicotiana benthamiana and fusion with a hydrophobin tail (see Figure 1). This fusion with hydrophobin allows for a more efficient and simplified purification of the recombinant protein, which represents a significant advantage over conventional methods. Hydrophobin has specific affinity properties with certain materials, facilitating the purification of recombinant protein in greater quantity and with greater purity (Biochem., 2020, v. 43, n. 37; Front. Plant. Sci., 2020, v. 10, n. 1674; BMC Biotec., 2014, v. 14, n. 1; PLoS ONE, 2014, v. 9, n. 6). [06] This approach presents important benefits, such as reduced production costs, process scalability, and the production of high-quality recombinant proteins. Furthermore, the use of the Nicotiana benthamiana plant platform offers a more accessible and sustainable alternative compared to traditional expression systems, such as animal or bacterial cells (Biochem., 2020, v. 43, n. 37; Front. Plant. Sci., 2020, v. 10, n. 1674; BMC Biotec., 2014, v. 14, n. 1; PLoS ONE, 2014, v. 9, n. 6). [07] The methodology described in the present invention utilizes cloning of the SARS-CoV-2 RBD domain gene sequence into a specific expression vector, pCAMGate, which is based on the CaMV 35S promoter. Expression occurs through transformation of Agrobacterium tumefaciens and infiltration into the leaves of Nicotiana benthamiana plants (see Figure 1). [08] Maceration of the leaves is done with liquid nitrogen, an extraction method that employs rapid cooling of plant cells followed by their rupture. The rapid immersion of the leaves in liquid nitrogen induces a thermal shock, forming ice crystals that break the cell walls. The frozen leaves are then crushed to release the desired intracellular components. This procedure preserves heat-sensitive compounds and results in a more effective extraction. [09] After extraction of the recombinant protein, purification occurs using an aqueous two-phase separation system (ATPS), which uses the surfactant Triton X-114. This method allows obtaining a semi-purified recombinant protein efficiently. [10] The present invention represents a significant advance over the prior art by offering a more efficient and simplified approach to the production and purification of recombinant RBD domain protein of the SARS-CoV-2 virus. Fusion with hydrophobin plays a key role in facilitating protein purification, resulting in greater purity and reduced process costs and complexity. This innovative methodology has the potential to boost research, therapy development, and vaccine production related to the virus, bringing economic, technical, and scientific benefits to the fight against COVID-19. [11] In the patent field, some relevant documents were located, which will be described below: document RU2752858C1 describes the creation of an integrative plasmid vector known as pVEAL2-S-RBD. This vector was designed to enable the efficient expression and secretion of the SARS-CoV-2 coronavirus RBD protein, but in mammalian cells. A polyhistidine tail is also incorporated for facilitated purification of the recombinant p