BR102017008691A2 - recombinant sugarcane cystatin for reducing erosive tooth wear and protection against tooth decay - Google Patents

Abstract

recombinant sugarcane cystatin to reduce erosive tooth wear and protect against tooth decay. describes the heterologous expression, purification and insertion in dental products of a recombinant sugarcane cystadine to reduce erosive tooth wear and protect against tooth decay called canecpi-5. which is of great importance in the dental segment due to its inhibitory action against cathepsin b and because it is an acid-resistant protein, when incorporated into the acquired tooth enamel film, it has the potential to protect the tooth structure from acid attacks of origin. microbial or not, because it prevents its dissolution and increases the resistance to erosive tooth wear.

Description

(54) Title: RECOMBINANT CYSTATINE OF SUGARCANE TO REDUCE EROSIVE DENTAL WEAR AND PROTECTION AGAINST DENTAL CARIES (51) Int. Cl .: C07K 14/415; C12N 15/29; A61K 8/64; C12N 15/66; A61P 1/02.

(71) Depositor (s): FOUNDATION UNIVERSIDADE FEDERAL DE SÃO CARLOS.

(72) Inventor (s): FLAVIO HENRIQUE DA SILVA; MARÍLIA AFONSO RABELO BUZALAF; ANDRÉA SOARES DA COSTA FUENTES; ADELITA CAROLINA SANTIAGO; JOHN MICHAEL EDWARDSON; MARIANA CARDOSO MIGUEL.

(57) Abstract: SUGAR CANE RECOMBINANT CYSTATINE TO REDUCE EROSIVE DENTAL WEAR AND PROTECTION AGAINST DENTAL CARIES. Describes the heterologous expression, purification and insertion in dental products of a sugarcane recombinant cystadine to reduce erosive tooth wear and protection against tooth decay, called CaneCPI-5. Which is of great importance in the dental segment due to its inhibitory action against cathepsin B and that because it is an acid-resistant protein, when incorporated into the acquired film of tooth enamel, it has the potential to protect the dental structure from acid attacks, microbial origin or not, to prevent its dissolution and to increase the resistance to erosive dental wear.

A B CAteaoi

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RECOMBINANT CYSTATINE OF SUGAR CANE FOR REDUCING EROSIVE DENTAL WEAR AND PROTECTION AGAINST DENTAL CARIES BRIEF DESCRIPTION [001] This patent application for an unprecedented “RECOMBINANT CYSTATIN OF SUGAR CANE FOR REDUCING SUGAR CANE IS PROCESSED” AND PROTECTION AGAINST DENTAL CARIES '' which refers to the heterologous expression, purification of a new sugarcane cystatin, called CaneCPI-5, and its insertion in dental products, due to its inhibitory action against cathepsin B and because it is an acid-resistant protein with the potential to protect the tooth structure from acid attacks, whether microbial or not; and to increase the resistance to erosive dental wear.

FIELD OF APPLICATION [002] The present invention belongs to the biotechnology section, more specifically to the dental area, as it deals with a product whose active component is an acid-resistant protein, with the potential to protect the dental structure from attack by acids, both microbial as non-microbial in origin, and erosive tooth wear.

CONVENTION [003] Food is essential for sustaining life, so that it is considered adequate, different types of food, with different nutrients and properties are eaten daily. Conventionally, the food process begins with the mouth, which has important characters for the digestive process, teeth and saliva. The consumption of some types of food favors dental wear, as well as poor hygiene of the teeth favors the proliferation of caries-causing bacteria. In this way, it can be said that eating habits are directly related to the two main types of dental injuries caused by acids: caries and erosive tooth wear.

[004] Although it can be easily prevented, dental caries is the most prevalent chronic disease worldwide, considered an important public health problem, it consumes considerable resources for its treatment and its sequelae.

[005] In the European Union, in 2011, annual spending on dental treatment was estimated at 79 billion euros (Rugg-Gunn 2013), while in the United States

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2/23 of America, this cost was $ 111 billion in 2012 (Wall, Nasseh et al. 2014). [006] Statistical data on dental treatment expenditures in Brazil are not easily available, but due to the economic development and the HDI (human development index) of some European countries and the United States of America, it can be speculated that figures proportionally approximate also affect Brazil. Worldwide, about 60-90% of children and almost 100% of adults have or have had cavities, which often leads to pain or discomfort (Organization 2012). In addition, dental diseases, including caries, are the main cause of the absence of children in school (Rugg-Gunn 2013). Data on the prevalence of erosive dental wear are more difficult to find, especially at the national or global level, not to mention the fact that the different registration systems make it difficult to compare the different studies. The few data available reveal an increase in prevalence, ranging from 1 to 79% in primary teeth of children aged 2 to 5 years, 14% in the permanent teeth of children aged 5 to 9 years, from 7 to 100% in adolescents aged 9 to 20 years and 4 to 100% in adults (18 to 88 years) (Jaeggi and Lussi 2014). A recent systematic review estimated the prevalence of erosive dental wear on permanent teeth of children and adolescents at 30.4% (Salas, Nascimento et al. 2015). It must be considered that erosive tooth wear is progressive and, if appropriate preventive measures are not taken in time, the prevalence tends to increase with age (Jaeggi and Lussi 2014). Since the prevalence of caries and erosive tooth wear is high and its undesirable sequelae, prevention is highly necessary.

[007] The main difference in the etiology of caries and erosive tooth wear is due to the fact that the acids involved in caries are produced by microorganisms established in a cariogenic biofilm (Takahashi and Nyvad 2011), while the acids involved in erosive lesions come from diet (extrinsic) (Barbour and Lussi 2014) or gastric content of the host (intrinsic) (Moazzez and Bartlett 2014). The pH of the acids involved in the etiology of these lesions is also different. In the case of caries, when the host has a high sugar diet, the microorganisms produce acids, reducing the biofilm pH below the critical for hydroxyapatite (pH 5.5), but staying above the critical for fluorapatite (pH 4.5 ), what

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3/23 ends up generating subsurface demineralization, clinically characterized as a white spot, which characterizes a non-cavitated lesion (Buzalaf and Whitford 2011). During this stage, it is still possible to reverse the process, avoiding cavitation and consequently the need for restoration (Stahl and Zandona 2007). In the case of erosion, although there is no characteristic fixed critical pH, as in the case of caries, the “critical pH” corresponds to a pH value in which the solution is exactly saturated in relation to a specific solid, such as enamel. This depends both on the solubility of the solid of interest and on the activities of the relevant mineral constituents of the solution, calcium, phosphate, and fluoride, to a lesser extent (Lussi and Carvalho 2014). In erosion, there is subsaturation in relation to both hydroxyapatite and fluorapatite, initially causing a softening of the tooth surface, followed by continuous dissolution of layer by layer of the enamel crystals, culminating in leading to a permanent loss of volume, with a softened layer. on the surface of the remaining tissue. In the more advanced stages, dentin ends up being exposed (Lussi, Schlueter et al. 2011). With dentin exposure, the progression of both the carious lesion and the erosion lesion undergoes a significant change due to the composition of the dentinal tissue.

[008] Due to the multifactorial etiology of both caries and dental erosion, several preventive and therapeutic possibilities have been proposed to control these injuries. These strategies are, in the first instance, focused on aetiological factors (Magalhães, Wiegand et al. 2009, Davies and Blinkhorn 2013, Rugg-Gunn 2013, Lussi and Hellwig 2014, Carvalho, Colon et al. 2015). Saliva is one of the most important biological factors involved in caries and erosion, having a protective role due to its different functions (Hannig and Balz 1999, Hannig and Balz 2001, Lussi, Jaeggi et al. 2004, Van Nieuw Amerongen, Bolscher et al. 2004, Hara, Ando et al. 2006). It dilutes acids and promotes their elimination from the oral cavity, through clearance-, has buffering capacity, neutralizing acids; it is supersaturated in relation to the dental mineral, providing calcium, phosphate and fluoride ions for remineralization; has a rich protein content, with different protective properties, in addition to contributing to the formation of the acquired film (Nicolau 2009, Buzalaf, Hannas et al. 2012), which is an organic layer, free of bacteria, which is formed in vivo as a result adsorption

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4/23 selective salivary proteins on the enamel surface (Dawes, Jenkins et al. 1963). Its formation is a dynamic process, influenced by several factors, such as circadian cycle, composition of the oral microbiota, proteolytic capacity of the oral environment and physical-chemical properties of dental surfaces, as well as location in the oral cavity (Lendenmann, Grogan et al. 2000) .

[009] The formation of the acquired film begins just a few seconds after the enamel is exposed to saliva, with a rapid increase in its thickness, reaching about 10-20 nm in the first minutes, which is stable for about 30 minutes (Hannig 1999). Initially, selective adhesion of precursor proteins occurs, with high affinity for hydroxyapatite, which interact electrostatically with the enamel surface (Hay 1973). Among them are proline-rich proteins (PRPs), staterin and histatins, as identified by in vitro studies, although in situ studies have also confirmed the presence of mucins, amylase, cystatines, lysozyme and lactoferrin (Siqueira, Custodio et al 2012).

[010] This first layer of proteins that adheres seems to be the one that gives the greatest protection against dental demineralization, since it is a very electron dense layer. Subsequent layers have a much looser arrangement when compared to that of basal films (Hannig 1999). It has been reported that there is no difference in the protective potential against demineralization of films formed after 3 minutes of exposure to saliva, when compared to those formed 2 hours later (Hannig, Fiebiger et al. 2004).

[011] In the second stage of film formation, called the maturation stage, there is a rapid increase in its thickness (100-1000 nm), which, together with the presence of globular structures, suggests the involvement of protein aggregates, more than individual proteins in their development (Hannig and Balz 2001).

[012] The thickness of the film reaches a plateau, between 30 and 90 minutes, with a thickness between 100 and 1000 pm, depending on its location in the oral cavity, being thicker in the buccal region when compared to the lingual (Hannig 1999). The film can still undergo intrinsic and extrinsic maturation, which can affect its solubility. Intrinsic maturation can be caused by the presence of transglutaminase, derived from oral epithelial cells, which can cross-link

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5/23 between basic PRPs and staterin (Yao, Lamkin et al. 2000, Hannig, Spitzmuller et al. 2008), as well as by the presence of alkaline phosphatase. In fact, enzymatic cross-linking and dephosphorylation seem to be the most important events for the intrinsic maturation of the film, while proteolysis appears to be of less importance (Hannig, Spitzmuller et al. 2008). Salivary proteolysis, which can occur before (Helmerhorst, Alagl et al. 2006) or after adsorption to hydroxyapatite (McDonald, Goldberg et al. 2011), plays an important role in the extrinsic maturation of the film, since many of its components they are peptide fragments (Vitorino, Calheiros-Lobo et al. 2007, Siqueira and Oppenheim 2009). The formation and maturation of the film can also be influenced by extrinsic factors, such as tooth whitening products, abrasive toothpastes and ingestion of acidic foods and drinks (Hara and Zero 2010).

[013] The incorporation of salivary proteins in the acquired film can affect its ability to protect against erosion. Erosion patients have half the amount of proteins in the acquired film formed in situ when compared to the control group, which reinforces the importance of the protective role of proteins present in the acquired film (Carpenter, Cotroneo et al. 2014). Among these proteins is mucin, which, when adhered to the enamel surface, alone or combined with other proteins, inhibits demineralization caused by erosive attack (Cheaib and Lussi 2011). In addition to mucin, staterins and proline-rich proteins (PRPs) are capable of maintaining a state of supersaturation in relation to calcium and phosphate in the oral cavity, by inhibiting their precipitation at neutral pH and releasing these ions after an acid attack and during demineralization. It has recently been reported that the calcium concentration in the film acquired in patients with erosion is reduced by 50% and the concentration of staterin (a calcium-binding protein) is reduced by 35% (Carpenter, Cotroneo et al. 2014). Regarding the proteins present in the diet, casein seems to have acid-protective properties (Barbour, Shellis et al. 2008), especially when combined with mucin (Cheaib and Lussi 2011). Similar to casein, ovalbumin can also reduce the dissolution of hydroxyapatite by acidic solutions in vitro (Hemingway, Shellis et al. 2008). In a recent study by a research group formed by researchers from UFSCar and USP,

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6/23 proteins that remain in the acquired film formed in situ on the dentin and in vivo on the enamel after exposure to citric acid or lactic acid, simulating erosive or carious processes, respectively. Acid resistant proteins were identified, such as mucins and cystatin B (Delecrode, Siqueira et al. 2015, Delecrode, Siqueira et al. 2015b).

[014] Some studies have reported important applications for recombinant cystatins, including in the field of dentistry. Cystatins are reversible protease inhibitor proteins, more specifically, cysteine-peptidases. Cystatins found in plants are called phytocystatins, and are a subfamily independent of the cystatine superfamily. Sugarcane cystatins were first described by Reis and Margis (2001), who identified in the SUCEST (Sugarcane Expressed Tags Project) database of sugarcane ESTs, twenty-five probable cystatines, which were classified into four groups through phylogenetic analysis. Recently, the Molecular Biology Laboratory of the Department of Genetics and Evolution at UFSCar identified and studied the activity of two new sugarcane cystatins, called Cane-CPI5 and CaneCPI-6. These proteins were able to inhibit the activity of human cathepsins and also of insect pests (Miguel, 2014).

[015] Based on the above, it has been suggested that the reinforcement of the acquired film by altering its composition, through “film engineering”, has been suggested as a promising measure to prevent dental demineralization (Vukosavljevic, Custodio et al. 2014). Within this concept, the search for acid-resistant proteins in the film, as well as for compounds that, once incorporated into the film can increase its acid-resistant potential, is extremely important. Once identified, these proteins or protective compounds can be incorporated into dental products, such as toothpaste, mouthwash solutions or gels, in order to enrich the film with them and, consequently, increase their acid-resistant capacity. Human cystatin B has been shown to be of interest for incorporation into dental products (Delecrode etal 2015b). However, the enzyme from humans has a high production cost. Thus, the use of Cane CPI-5 for this purpose is highly promising.

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BACKGROUND OF THE INVENTION [016] In the current state of the art, some documents are described that describe the use of cystatine group proteins as active components of dental compositions. Among the previous findings, none mentioned the use of a heterologous protein from sugar cane in the composition of the products and / or processes described. Thus, none of the previous findings fully anticipates the proposal described by this patent application.

[017] In the current state of the art is the previous PI9307800-5, entitled Anti-caries compositions that describe oral and edible compositions that offer anti-caries benefits, whose primary active ingredient is a protein, in its entirety or fragments of it, selected from the group constituted salivary proteins, including cystatin S, cystatin SA, cystatin SN and their mixtures.

[018] Previously US2002052476, Disclosed is a human CysE polypeptide and DNA (RNA) encoding such polypeptide, which describes a process for obtaining recombinant techniques for a polypeptide, called CysE, its DNA and Coding RNA and its use in the treatment of osteoporosis, tumor metastasis, microbial infections, viral infections, septic shock, inflammation, retinal irritation, caries, cachicia and muscle loss; the foregoing also mentions the use of CysE in methodologies for detecting mutations in its coding region and altering the concentration of polypeptides in a sample derived from a host. Although the mentioned protein is used against caries, its origin is different from that proposed in this patent application, CaneCPI-5, whose origin is sugarcane, in addition to being quite different in sequence of amino acids, only 23 of identity in 125 aligned. The proposition of using human cystatin for caries is related to its antimicrobial potential, because it inhibits cysteine peptidases essential for microorganisms in bacterial biofilm, CaneCPI-5, in addition to this property, also presents a strong interaction with dental enamel, Its mechanism of action is related to its incorporation in the acquired enamel film, in order to prevent the enamel dissolution, both by acids of microbial origin (acting against tooth decay) and by acids of non-microbial origin (acting against dental erosion. ); what evidence

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8/23 that the action of CaneCPI-5 against caries is more efficient than that of human recombinant cystatin, since it has two mechanisms of action, inhibition of microorganisms and binding to enamel, which human cystatin does not have.

[019] From the above, it is observed that none of the previous findings fully anticipates the proposal of the present patent application. Thus, none of the foregoing anticipates the proposal that describes the heterologous expression, purification of a sugarcane recombinant cystatin and its insertion in dental products to reduce erosive tooth wear and protection against dental caries.

OBJECTIVE OF THE INVENTION [020] The present invention aims to provide a product that provides significant protection from the mineral loss of tooth enamel, through its incorporation into the acquired film of tooth enamel, in order to prevent the enamel dissolution by both acids of origin microbial, acting against dental caries, and by acids of non-microbial origin, acting against dental erosion.

OF THE INVENTION [021] The present patent application describes the heterologous expression, purification of a sugarcane recombinant cystatin, called CaneCPI-5, and its insertion in dental products to reduce erosive tooth wear and protection against caries dental. Which is of great importance in the dental segment due to its inhibitory action against cathepsin B and because it is an acid-resistant protein that, when incorporated into the acquired film, has the potential to protect the dental structure from acid attacks, of microbial origin or not; and, it increases the resistance to erosive tooth wear.

ADVANTAGES OF THE INVENTION [022] In short, the present invention has as main advantages:

J Provide a product with the potential to protect the dental structure from acid attacks, whether microbial or not, and to increase resistance to erosive dental wear;

J Provide a product with protective paper against demineralization of tooth enamel;

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J Have a product to be incorporated into dental products, such as toothpaste, mouthwash solutions, gels for topical application, chewable tablets, chewing gum and mouth sprays;

J Have a product with a low production cost and a relatively simple production process.

DESCRIPTION OF THE FIGURE [023] The invention will be described in a preferred embodiment, thus, for better understanding, references will be made to the flowchart and the attached images.

J Figure 1: Flowchart of the process for obtaining CaneCPI-5;

J Figure 2: ORF amplification of sugarcane cystatin. The CaneCPI-5 cystatin gene region was amplified by PCR from clone SCVPAM1059C07.g, using specific primers. In the gel columns: (L) 1 Kb DNA ladder (Invitrogen), (1) negative PCR control, and (2) PCR product corresponding to cystatin ORF. bp: base pairs. The arrow indicates the amplified band of approximately 345 bp for CaneCPI-5. 1% agarose gel.

J Figure 3: Heterologous expression and periodic collection every 1 hour after induction of cystatin CaneCPI-5. SDS-PAGE analysis 15% of samples collected from soluble (1), and insoluble (2), non-induced (3) fractions, with 1 hour of induction (4), with 2 hours of induction (5), with 3 hours of induction (6), and with 4 hours of induction (7). M: BenchMark ™ Protein Ladder (Invitrogen). The arrow indicates the band referring to the cystatin CaneCPI-5 (-15.2 kDa).

J Figure 4: Heterologous expression and purification of the CaneCPI-5 cystatin. SDS-PAGE analysis 15% of samples collected from non-induced (1), and induced (2), insoluble (3), and soluble (4) fractions, resulting from cell lysis, and nickel column purification ( 5). M: BenchMark ™ Protein Ladder (Invitrogen). The arrow indicates the band referring to the cystatin CaneCPI-5 (-15.2 kDa).

J Figure 5: Inhibition of human cathepsin B by recombinant cystatin. The inhibitory activity of cystatin CaneCPI-5 against the human cathepsin B enzyme was measured in a fluorometric assay using the substrate Z-Phe-Arg-MCA. The graph represents the inhibitor inhibition curve, obtained by calculating the residual activity of cathepsin B

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10/23 human as the inhibitor was added. Values are expressed as the mean ± the standard deviation of three independent experiments.

J Figure 6: AFM images typical of enamel samples, which were subjected to various treatments. (A) Control (untreated). The arrows indicate lines resulting from polishing. (B) Citric acid. (C) Mucina. (D) Casein. (E) Mucine plus casein. (F) CaneCPI-5. The color scale indicates 0-23 nm for (A), (B) and (E), and 0-25 nm for (C), (D) and (F).

J Figure 7: Typical force curves. (A) Control (without protein). (B) Mucina. (C) Casein. (D) Canacystatin-5. Attention to the difference in the force scale between (AC) θ (D).

J Figure 8: Average change in percentage of hardness after 1 or 3 days of treatment of enamel samples with human saliva for 2 hours, then with protein solutions for 2 hours, followed by challenge with 0.65% citric acid for 1 minute. The treatment was carried out once / day for 3 days.

[024] DESCRIPTION OF DNA AND PROTEIN SEQUENCES

J Seq ID no 1: CaneCPI-5 Forward primer

J Seq ID no 2: CaneCPI-5 Reverse primer

J Seq ID no 3: CaneCPI-5 coding sequence (open reading frame - ORF);

J Seq ID no 4: Complete CaneCPI-5 amino acid sequence translated from the ORF described in Seq ID no 3;

J Seq ID no 5: CaneCPI-5 coding sequence, without the signal peptide, cloned in plasmid pET28a;

S Seq ID # 6: CaneCPI-5 final DNA sequence, including additional bases (each of histidines and others) of plasmid pET28a;

J Seq ID no 7: Amino acid sequence of CaneCPI-5 cloned in pET28a, expressed and used in the experiments described in the present application.

DETAILED DESCRIPTION OF THE INVENTION [025] The process of obtaining RECOMBINANT CYSTATINE FROM SUGARCANE TO REDUCE EROSIVE DENTAL WEAR AND PROTECTION

AGAINST DENTAL CARIES (Figure 1) begins with the amplification (E1) of the coding region (1) of CaneCPI-5, Seq ID no 3, by polymerase chain reaction

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11/23 (Polymerase Chain Reaction - PCR) from the cDNA clone SCVPAM1059C07.g (Project SUCEST-FAPESP), using the primers described in Seq ID no 1 and Seq ID no 2, using the restriction enzymes Nhe- \ e BamH- \ (Thermo Fisher Scientific), for further cloning into the expression vector pET28a (Novagen). The amplification (E1) was performed in a final volume of 25 μL, containing 30 ng of mold, 200 μΜ of each dNTP (Promega), 1x reaction buffer (Tris-HC1100 mM pH 8.8, MgCI2 16 mM, KCI 500 mM), 10 pmoles of each primer and 1.25 U of the Taq DNA polymerase enzyme (SINAPSE Biotecnologia Ltda.). For amplification (E1), the T100 Bio-Rad thermocycler was used with the following amplification cycle: denaturation 94 ° C for 3 minutes, 35 amplification cycles at 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 60 seconds and a final extension at 72 ° C for 10 minutes. The amplified product (2) was analyzed (E2) on a 1% agarose gel, containing 1 pg / mL of ethidium bromide and visualized (E3) in UV on the ChemiDoc MP System Bio-Rad equipment.

[026] For divination (E4) with the enzymes (3) Nhe \ and BamHI (1.5 U), 2 pg of the amplified product (2) and the vector pET28a (4) were used in a 50 pL reaction at 37 ° C for 2 hours. The cleaved products (5) were recovered (E5) on 1% agarose gel and purified (E6) with the Wizard SV Gel and PCR Clean Up System kit (Promega). For subsequent binding (E7), 50 ng of the vector pET28a (4) and 16 ng of the insert were used using 1 U of the enzyme T4 DNA ligase (Thermo Fisher Scientic) (6) in a final reaction volume of 10 pl_, incubated ( E8) at 4 ° C for 16 hours. The bond was transformed (E9) into chemocompetent E. coli DH5a. After transformation (E9), the bacteria (7) were plated (E10) in LB selective medium containing 25 pg / mL of the kanamycin antibiotic and incubated at 37 ° C for 16 hours. The recombinant colonies (8) were confirmed by PCR (E11) using the primers and reaction previously mentioned.

[027] For recombinant expression, the vector pET28a with CaneCPI-5 (8) was transformed (E12) into chemocompetent bacteria E. coli Rosetta (DE3) (9). The transformed bacteria (9) were plated (E13) in LB medium containing 25 pg / mL of the antibiotics kanamycin and chloramphenicol and incubated (E14) at 37 ° C for 16 hours. A recombinant colony (10) was isolated (E15) for the pre-inoculum in 5 ml of LB culture medium with 25 pg / ml of kanamycin and chloramphenicol and the culture (11) was kept under agitation (E16) at 200 rpm at 37 ° C for 16 hours in New Brunswick Scientific IL shaker

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Ecxella E24. After this period, the pre-inoculum (12) was added (E17) to 500 mL of LB medium containing 25 pg / mL of kanamycin and chloramphenicol under agitation (E18) at 200 rpm at 37 ° C until reaching ϋΟβοο of 0, 5. The expression (E19) of CaneCPI-5, Seq ID no 6, was induced by the addition of IPTG (Isopropyl β-Dl-thiogalactopyranoside) (13) at a final concentration of 0.4 mM for 4 hours under the same stirring conditions (E20) and temperature, from 200 rpm to 37 ° C. After the induction period, the cells (14) were centrifuged (E21) in a Sorvall RC-5C centrifuge at 5500xg, 4 ° C for 10 minutes. The precipitate (15) was resuspended (E22) in 30 ml of lysis buffer (100 mM NaCI, 50 mM NahhPCU, 10 mM Tris), pH 8.0 and sonicated (E23) with five one-minute pulses at the power of 20 %, at 30-second intervals, using the Sonic Dismembrator 500 (Fisher Scientific) sonicator. The lysate (16) was centrifuged (E24) at 11900 xg, 4 ° C for 15 minutes and the supernatant (17), after filtering (E25) on 0.45 pm Millipore membrane, was purified (E26) by affinity chromatography, a column containing 3 ml of Ni-NTA Superflow nickel resin (Qiagen) (18) was used to purify CaneCPI5 (17), Seq ID no 7. The column was equilibrated with 5 volumes of lysis buffer (19) and the supernatant containing CaneCPI-5 (17) was applied. The resin (20) was washed (E27) with 3 volumes (9 ml) of lysis buffer (19). The protein (21) was eluted (E28) with 2 volumes (6 ml) of lysis buffer (19) containing different concentrations of imidazole (10, 25, 50, 75, 100 mM), and 3 volumes (9 ml) containing 250 mM imidazole. The eluted fractions (22) collected were analyzed (E29) on denaturing polyacrylamide gel (sodium dodecyl sulfate polyacrylamide gel electrophoresis - SDS-PAGE) 15%. The purified protein (23) was dialyzed (E30) using a 3500 MWCO membrane (Pierce) against 1 liter of PBS buffer (24) pH 8.0, at 4 ° C, making three changes (of 1 liter): one after 1 hour , one after 2 hours and one after 16 hours. The CaneCPI-5 (25) was then sterilized (E31) in a Millex-GV 0.22 pm filter (Millipore) and its concentration was determined (E32) by the Bradford method, 1976, using Hitachi U5100 Spectrophotometer and the CaneCPI-5 ready for used (26) has been made available. The product (26) can be immediately added (E33) to dental products or stored (E34) in a freezer -20 ° C (or 80 ° C), indefinitely. CaneCPI-5 ready (26) can still be lyophilized (E35) for stabilization, preferably with cryostabilizers such as glycerol, sorbitol, polyethylene glycol, xylitol, glucose, among others.

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13/23 [028] In addition to the vector pET28a and its transformation into chemocompetent E. coli DH5a, other expression systems such as filamentous fungi and yeasts, including Pichia pastoris, can be used. The protein could be produced in transgenic plants, such as sugar cane, tobacco, Arabidopsis, tomato etc. The synthesis could still be performed independently of biological systems, that is, in vitro. Although they have not yet been tested, other cystatines from sugar cane may also have dental properties similar to CaneCPI-5 (26), object of the present patent application.

[029] The CaneCPI-5 (26) due to its properties can be added to dental products such as toothpaste, gels, mouthwash solutions and even chewable tablets, chewing gum and mouth sprays, in an amount that varies between 0.05 to 3% w / w, according to the product. Thus, some formulations for these products are proposed, which are non-limiting examples of the invention:

[030] The dentifrice has in its composition abrasive components, between 4 to 50% w / w, according to the degree of abrasiveness desired, among them calcium carbonate, calcium pyrophosphate (conditioned to the use of monofluorophosphate as a fluorinated agent) or, preferably , silica gel; humectant components, ranging from 20 to 65% w / w, which may be sorbitol, glycerin, polyethylene glycol, propylene glycol and / or mixtures thereof; binding components, between 0.5 to 7% w / w, which may be carboxymethyl cellulose, xanthan gum, thickened silica or mixture thereof; surfactant components, between 1 to 6% w / w, which may be betaines, cocoamidopropyl betaine or, preferably, sodium lauryl sulfate or mixture thereof; preserving agents, between 0.01 and 0.5% w / w, which may be methylparaben, propylparaben, sodium benzoate, methyl hydroxybenzoate or mixture thereof; fluoridated agents, between 500 to 5000 ppm, which may be sodium monofluorophosphate, titanium tetrafluoride, stannous fluoride, amine fluoride, preferably sodium fluoride, or a mixture thereof; antiplaque agents, which can be chlorhexidine, cetylpyridine chloride, triclosan and CaneCPI-5 (26), between 0.05 to 3% w / w. The toothpaste formulation described has an acidic pH ranging from about 6.0 to 7.0.

[031] The dental gel composition comprises hydroxycellulose as an adjuvant component; as a binding agent glycerin, more specifically; O

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14/23 propylene glycol as a wetting component, in addition to methyl paraben and imidazolidinyl urea as preservatives and deionized water in a maximum amount of 7.5%. The concentration of CaneCPI-5 can vary from 0.05 to 3%. Optionally, if the amount of water is exceeded, an antioxidant, retinoic acid, a vitamin A acid is added.

[032] The mouthwash solution will be prepared with deionized water, flavoring, preservative (methylparaben and imidazolidinyl urea) and CaneCPI-5 (26) with a concentration varying between 0.05 and 3%.

[033] The following are the results of tests performed with CaneCPI-5 (26):

[034] The potential of CaneCPI-5 (26) to bind to dental enamel and protect against acid dissolution was analyzed using Atomic Force Microscopy, from the one described below.

Sample preparation [035] The samples were cut from the middle of the crown face of the crowns using a low speed ISOMET saw (Buehler, Lake Bluff, IL, USA) with two diamond discs (Extec, Enfield, CT, USA) separated by a 4 mm and thick spacer. The enamel surface was ground with water-cooled silicon carbide discs (320, 600 and 1,200 quality documents, ANSI grain; Buehler) and polished with wet filter paper with diamond spray. Finally, the samples were cleaned in distilled water using an ultrasound bath to remove any residue.

[036] Measurement of the protein enrichment effect of the acquired film on the change in surface hardness in response to acid [037] Enamel samples were divided into five groups (n = 15 for each group): water (control), mucin (2.7 mg / ml) plus casein (5 mg / ml), cystatin-B (0.025 mg / ml), CaneCPI-5 (0.025 mg / ml), and CaneCPI-5 (0.025 mg / ml) applied before formation of the acquired film. Stimulated saliva was collected from three volunteers and used to form an enamel film acquired over the samples for 2 hours. Samples from the first four groups were exposed to protein solutions with stirring at 30 ° C for 2 hours. For the final group, the enamel samples were treated with CaneCPI-5 in solution before the film was formed. All samples were then incubated in 0.65% citric acid (pH 3.4) for 1 minute

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15/23 to 30 ° C. The treatment was conducted once a day for three days. To measure the hardness of the enamel surface, five notches were made, using 25 g for 10 seconds (Knoop-KHN, kgf / mm ² ; Shimadzu HMV-2 Tokyo, Japan Micro hardness tester) and the percentage hardness variation was calculated. surface (% SHC). The data were analyzed by ANOVA and Tukey's test (p <0.05).

[038] Measurement of the effect of the protein layer on the surface roughness, in response to acid [039] Enamel samples were divided into five groups (n = 8 for each group): control (without protein), mucin (2.7 mg / ml), casein (10 mg / ml), mucin (2.7 mg / ml) plus casein (10 mg / ml), and CaneCPI-5 (0.85 mg / ml). All samples were previously washed with Milli-Q water and dried in a stream of nitrogen gas. The samples were mounted on steel discs with double-sided tape and digitized in the air using a Bioscope atomic force microscope controlled by a Nanoscope lla (Bruker, Coventry, United Kingdom). The microscope was operated in touch mode using silicon beams (OTESPA-R3, Bruker, UK) with a spring constant of 26 N / m and a resonance frequency of 300 kHz. For protein treatment, the enamel samples were then incubated in solutions containing the protein concentrations described above for 4 hours at room temperature, with gentle agitation. [040] The samples were rinsed with water and dried. Atomic force microscopy (AFM) was conducted to detect any change in surface roughness as a result of protein deposition. All samples (control and protein treated) were then incubated in a 0.65% citric acid solution (pH 3.5) for 1 minute at room temperature with gentle agitation, and washed again with water and dried. Images of the samples were obtained again to assess the effect of the acid on the surface roughness. AFM images were leveled using the Nanoscope software and processed using the Gwyddion 2.44 free scanning probe microscopy data analysis Software (http://gwyddion.net/). Specifically, eight lines of diagonal profiles (128 pixels wide) were drawn randomly in each image, to obtain the average roughness value for the image. The roughness values for the different groups were compared using an unpaired t test, with a significance level of 0.05.

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Measurement of molecular strength by AFM [041] Enamel samples were washed with Milli-Q water and dried in a nitrogen gas stream. The samples were mounted on steel discs, using Azul Tack®, and the discs were fixed, using BluTak®, in the center of 6 cm Petri dishes. Phosphate-buffered saline (pH 7.4; 4.5 ml) was added to the plate so as to submerge the enamel sample. Mucin, casein and CaneCPI-5 were covalently coupled to AFM silicon nitride tips (the MLCT probe cantilever C (Bruker), which had an excitation frequency of 7 kHz and a specified spring constant of 0.01 N / m), via a flexible polyethylene glycol linker (PEG18).

[042] Specifically, the tips were amino-functionalized using 3aminopropyltriethoxysilane (APTES, and then reacted with acetal-PEG18-NHS. The aceto groups were converted to aldehyde groups by incubating the tip in 1% citric acid, followed by washing in water Then, the aldehyde groups reacted with amino groups of the protein by incubating the tip with a solution of the protein.

[043] Force measurements were performed under fluid at a pH of 7.4 with a Bioscope atomic force microscope (Bruker). Breaking forces and detachment separation values were obtained from force-extension curves, using the image processing Scanning Probe software.

[044] The potential of CaneCPI-5 (26) to bond to tooth enamel and protect against acid dissolution was also analyzed through functional Experiments, starting from the one described below.

[045] Preparation of enamel samples [046] Freshly extracted bovine incisors were used. The teeth were stored in 0.1% buffered thymol solution (pH 7.0) at 4 ° C (Kato et al. 2010). The crowns were separated from the roots with a diamond saw (Isomet 1000; Buehler, Lake Bluff, IL, USA), using two parallel diamond discs separated by a 4 mm spacer. 1-2 blocks were cut from the crown of each bovine incisor. The samples were individually fixed on acrylic discs with wax. The enamel surfaces of the blocks were leveled with water-cooled carborundum discs (320, 600 and 1200 types of AI2O3 paper; Buehler, Lake Bluff, IL, USA)., And polished with felt with diamond spray (1 micron; Buehler, Lake Bluff, IL, USA), on a machine

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17/23 rotary polishing. After polishing, the specimens were cleaned in an ultrasound bath (T7 Thornton, single Ind e with Ltda, São Paulo, SP, Brazil) for 7 minutes using deionized water.

[047] For the purposes of normalization, a pre-selection of the blocks was carried out based on their initial hardness. Six notches in the central region of the blocks (100 distances from each other) were made using a 50 g load (Knoop penetrator) for 15 seconds (HMV-2000; Shimadzu Corporation, Tokyo, Japan). The baseline values of surface hardness (SHb) were determined by the average of six cavities. Only samples with a 10% baseline indentation above or below the average were used. Saliva collection [048] The study protocol was approved by the Ethics Committee of the Faculty of Dentistry of Bauru, University of São Paulo. The volunteers donated saliva after signing a consent form. Samples of saliva stimulated in paraffin from 3 healthy donors of both sexes, aged 20-35 years, were collected in ice-cooled flasks and grouped. Collections were made between 9 and 11:00 am. Immediately after collection, all saliva was centrifuged for 20 minutes at 4 ° C and 14000 g. The supernatants were divided into 13 ml aliquots and stored at -80 ° C until use. The supernatants were collected with a pipette to form a pool.

Protein solutions [049] The following protein solutions were prepared, all prepared immediately before the experiments and their pH was adjusted to 7.0:

S 0.27% mucin + 0.5% casein: it was prepared using bovine milk casein (Sigma) and gastric pig mucin (Sigma) dissolved in deionized water.

S 0.025 ug / ul of cystatin B: it was prepared using recombinant human cystatin B (Sigma) dissolved in deionized water.

S 0.025 pg / μΙ CaneCPI-5: it was prepared using CaneCPI-5 dissolved in deionized water.

Groups and treatments

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18/23 [050] The specimens were selected according to their initial hardness and divided into 5 experimental groups (n = 15) according to the treatment received: 1) deionized water (control), 2) 0.27% mucin + 0.5% casein solution, 3) 0.025 g / uL cystatin-B solution, 4) 0.025 ug / uL CaneCPI-5 solution, and 5) 0.025 ug / uL CaneCPI-5 solution applied before film formation acquired. Except for group 5, the specimens were immersed in the prepared saliva (300 ml), for 2 hours at 30 ° C, under constant agitation. They were then rinsed in deionized water and exposed to the protein solution (100 mL) for an additional 2 hours at 30 ° C under constant agitation {Cheaib, 2011 # 2; Yin, 2006 # 3}. Group 5 samples were first immersed in the protein solution for 2 hours at 30 ° C under constant agitation, then rinsed in deionized water and exposed to the prepared saliva (300 mL) for an additional 2 hours at 30 ° C under constant agitation . The samples were washed once more in deionized water and were incubated in a 0.65% citric acid solution (pH 3.5) for 1 minute at 30 ° C under constant agitation (1 ml per sample). They were then rinsed with deionized water and air dried. Each sample was treated once a day for 3 days. The specimens were stored in a humidity chamber at 4 ° C between treatments.

Surface hardness measurement [051] Hardness measurements were performed using a Knoop diamond under a load of 50 g and a time of 15 seconds (HMV-2000; Shimadzu Corporation, Tokyo, Japan). Six notches were made in the center of the block, 100 distant from each other. Control notches of 2 and 5 g were made to detect the possible loss of surface {Cheaib 2011 # 2}. Surface hardness was analyzed at baseline and then on days 1 and 3 (SHT) and the percentage of change in surface hardness (% SHC) was calculated as a measure of enamel softening, according to the following equation:% SHC = [(SHB-SHT) / SHb] * 100.

Statistical analysis [052] GraphPad InStat software version 3.0 for Windows (GraphPad Software RIPC.,

La Jolla, CA, USA) was used. Since the assumption of normality (test of

Kolmogorov-Smirnov) and homogeneity (Bartlett's test) were satisfied, the data were analyzed by ANOVA and Tukey's test (P <0.05).

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Results obtained from the tests [053] Amplification of the ORF encoding the CaneCPI-5 cystatin and construction of the expression vector [054] The ORF was obtained by PCR, Seq ID no: 3, and the analysis of the amplification product in electrophoresis of agarose gel (Fig. 2) showed the presence of the fragment corresponding to cystatin in the expected size, approximately 345 bp. The PCR product (Fig.2) was linked to the propagation plasmid pTZ57R / T. Like other sugarcane cystatins, CaneCPI-1 (SOARES-COSTAetal., 2002), CaneCPI-2 and CaneCPI-3 (GIANOTTI et al., 2006), had already been efficiently expressed and purified using the vector pET- 28a, the same strategy was adopted in this study to obtain the CaneCPI-5 protein.

Heterologous expression and purification of recombinant cystatin [055] The ORF corresponding to cystatin was cloned into the expression vector pET-28a. This vector allows for directional cloning of the gene of interest in phase with a sequence, which encodes a six tail “histidine” at the C or N-terminus. This "HisTag" facilitates the purification of the fusion protein by affinity chromatography and can later be removed, if necessary, since between the protein sequence of interest and the histidines there is a site for the thrombin protease. Thus, the protein, CaneCPI-5 was expressed in fusion with the histidine tail (HisTag), in the N-terminal region, derived from the vector pET-28a, Seq ID no: 6. Considering that histidine residues have an affinity for nickel, it was possible to purify it in a single step by this procedure. The analysis in SDS-PAGE 15%, of the samples collected periodically every 1 hour, after the beginning of the induction, revealed that from the first hour, the recombinant protein was already expressed, and that there is not much difference in the expression levels of the samples collected in 2, 3 and 4 hours (Fig 3).

[056] The soluble and insoluble fractions resulting from the cell lysis of the bacteria showed that the cystatin CaneCPI-5 is mostly found in the soluble fraction. The soluble fraction was used for the purification of recombinant cystatin by affinity chromatography on a column containing nickel resin. This fraction was then added to the column and the protein was eluted in lysis buffer containing imidazole. Imidazole, which also has a strong affinity for nickel, competes with histidines for binding to

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20/23 this metal, releasing the fusion protein. The analysis on SDS-PAGE revealed that most of the CaneCPI-5 protein was eluted in the fractions that contained 250 mM imidazole (Fig 4). The yield of purified cystatin was approximately 16 mg per liter of cell culture.

[057] Inhibition of human cysteine peptidase cathepsin B activity by the new recombinant cystatin CaneCPI-5.

[058] The inhibitory activity of recombinant CaneCPI-5 against the enzyme cathepsin B, was measured in a Hitachi F2000 spectrofluorimeter, using the fluorogenic substrate Z-PheArg-MCA. The result, summarized in Figure 5, shows that the recombinant cystatin CaneCPI-5 showed inhibitory activity against cathepsin B.

[059] The value of the inhibition constant (Ki), also called the dissociation constant of the enzyme-inhibitor complex, expresses the affinity of the inhibitor for the enzyme (CHENG and PRUSOFF, 1973). For an inhibitor to be considered potent, its affinity for the target enzyme must be so high that its K \ value is less than or equal to the total enzyme concentration used in the inhibition assay (COPELAND, 2005). Considering the K value, found for the inhibition of cathepsin B, we can verify that the cystatin CaneCPI-5 showed a considerable inhibitory activity against this enzyme with K \ in the value of 6.87 nM.

[060] The ability to inhibit the enzymatic activity of cathepsins, in addition to other cysteine-peptidases, suggests that the recombinant cystatin CaneCPI-5 is in its correct conformation. The results obtained in this study reveal that the protein CaneCPI-5 was able to efficiently inhibit the activity of human cathepsin B, with a Ki similar to that of CaneCPI-4 (GIANOTTI etal., 2008).

[061] When compared to other cysteine-peptidases, cathepsin B contains a structural element, called an occluding loop, which blocks the cleft of the active site (MUSIL et al., 1991). As cystatins inhibit cathepsins through competitive binding to the active site, the conformation of this loop in cathepsin B is incompatible with the binding of cystatins. This influence of the loop on accessibility to the active site is reflected in high Ki values for most cystatins, when compared to those obtained against cysteine-peptidases that lack such a loop.

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21/23 (BARRETT etal., 1986; KONDO et al., 1990; ABRAHAMSON, 1994; OHTSUBO et al., 2005; MARTINEZ etal., 2005b).

[062] Effect of protein enrichment of the acquired film on changing surface hardness in response to acid [063] Enamel samples were incubated with saliva enriched with water (control), mucin (2.7 mg / ml) plus casein ( 5 mg / ml), cystatin B (0.025 mg / ml), and CaneCPI-5 (0.025 mg / ml) at 30 ° C for 2 h. In addition, a group of enamel samples was incubated with CaneCPI-5 before treatment with saliva. All samples were then incubated once a day for three days in 0.65% citric acid (pH 3.4) for 1 minute at 30 ° C. After one day, the SHC% for the control group was 46.9 ± 6.7 (SEM)%. Treatment with cystatin B (35.1 ± 9.9%) and CaneCPI-5 (35.2 ± 6.6%) before film formation significantly (P <0.05) significantly reduced% SHC compared to the control , while mucin plus casein and CaneCPI-5 during film formation had no effect. After three days, all cystatine treatments (54.5 ± 8.6, 55.5 ± 10.7 and 53.1 ± 9.3% for cystatin-B, and CaneCPI-5 during and before the formation of film, respectively) significantly reduced the% SHC compared to the control (67.6 ± 9.4%). In addition, treatment with CaneCPI5 before film formation significantly reduced% SHC. In contrast, the combination of mucin plus casein does not provide significant protection (64.4 ± 9.4%).

Effect of protein coating on surface roughness in response to acid:

[064] AFM topographic images (Fig.6) of untreated enamel samples and with samples coated with proteins: mucin (2.7 mg / ml_), casein (10 mg / ml), mucin (2.7 mg / ml) plus casein (10 mg / ml), and Canecystatin-5 (0.86 mg / ml), before and after incubation with citric acid (0.65%, pH 3.4, for 1 min), were carried out in air. As shown in Figure 6A, a typical untreated sample had a relatively smooth surface with a number of linear Scour lines (arrows). The average roughness of the untreated enamel was 0.28 ± 0.03 nm (n = 8). Images of enamels that had been exposed to citric acid showed a dramatic increase in surface roughness, even after a 1-minute treatment (Figure 6B), and the average roughness increased to 1.06 ± 0.05 nm (P <0 , 0001). Incubation of samples

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22/23 with proteins caused a deposition of visible material on the enamel surfaces. [065] This deposition reflected an increase in the average enamel roughness values treated with protein: 0.42 ± 0.04 nm for mucin, 0.40 ± 0.04 nm for casein, 0.33 ± 0.04 nm for mucin plus casein, and 0.46 ± 0.06 nm for CaneCPI-5). The protein-treated enamel values after citric acid treatment were: 0.93 ± 0.07 nm for mucin, 1.10 ± 0.10 nm for casein, 0.78 ± 0.09 nm for mucin plus casein, and 0.79 ± 0.06 nm for CaneCPI-5 (all values of n = 8). Of the three proteins, the only one that significantly protected against citric acid-induced enamel damage was CaneCPI-5 (P = 0.005).

Single strength measures of AFM molecule:

[066] Next, the adhesion of the three proteins mucin, casein and CaneCPI-5 was quantified, at the level of a single force molecule by spectroscopy using an atomic force microscope. The proteins were covalently coupled to the silicon nitride AFM probe through a flexible polyethylene glycol ligand (PEG18), in order to allow the protein to interact with the enamel surface in several orientations. Typical force curves for control discs (without any protein) and tips coupled to the protein are shown in Figure 7. Note that the control force, mucin and casein axes (Figures 7A-C), respectively, are identical to each other. others, but different from the force axis for CaneCPI-5 (Figure 7D). The detected forces were 0.177 ± 0.031 nN for the control, 0.221 ± 0.058 nN for mucin, 0.261 ± 0.050 nN for casein, and 0.933 ± 0.108 nN for CaneCPI-5 (n = 40 for each condition). Corresponding detachment separation values were 132.9 ± 14.9 nm for the control, 82.1 ± 10.8 nm for mucin, 92.5 ± 9.5 nm for casein, and 134.6 ± 10.1 nm for CaneCPI-5 (n = 40). [067] Thus, CaneCPI-5 demonstrated a high strength of the interaction with the enamel (and statistically significant; P 0.0001 <). In contrast, the forces shown by mucin and casein were not significantly different from the control.

Functional experiments [068] On day 1, treatment with cystatin B (35.1 ± 9.9%) and CaneCPI-5 (35.2 ± 6.6%) before film formation significantly reduced the% SHC compared with the control (46.9 ± 6.7%) ( Figure 11 A, P <0.05). On day 3, all cystatine treatments (54.5 ± 8.6,

55.5 ± 10.7 and 53.1 ± 9.3% for cystatin -B, CaneCPI-5 and CaneCPI-5 before formation of

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23/23 film, respectively significantly reduced the% of SHC compared to the control (67.6 ± 9.4%) (P <0.01). In addition, treatment with CaneCPI-5 prior to film formation significantly reduced the% of SHC compared to the mucin / casein combination (64.4 ± 9.4%) (Figure 8B, P <0.05).

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Claims (7)

1) SUGAR CANE RECOMBINANT CYSTATIN FOR REDUCING EROSIVE DENTAL WEAR AND PROTECTION AGAINST DENTAL CARIES, where recombinant cystatin (26) after fusion in the plasmid and insertion of the histidine sequence is characterized by encoding the final protein sequence to Seq ID # 6. 2) SUGARCANE RECOMBINANT CYSTATIN FOR REDUCING EROSIVE DENTAL WEAR AND PROTECTION AGAINST DENTAL CARIES, according to claim 1, where CaneCPI-5 (26) cloned in pET28a is characterized by having the following sequence of amino acids ID # 7. 3) SUGARCANE RECOMBINANT CYSTATIN FOR REDUCING EROSIVE DENTAL WEAR AND PROTECTION AGAINST DENTAL CARIES, according to claims 1 and 2, Cane CPI-5 (26) is characterized by being added to dental products in a proportion between 0.05 and 3% pp of the product. 4) SUGARCANE RECOMBINANT CYSTATINE FOR REDUCING EROSIVE DENTAL WEAR AND PROTECTION AGAINST DENTAL CARIES, according to claim 3, where the dental products to which Cane CPI-5 is added are characterized by being toothpaste, gels, mouthwash solutions, chewable tablets, chewing gum oral spray. 5) SUGAR CANE RECOMBINANT CYSTATINE FOR REDUCING EROSIVE DENTAL WEAR AND PROTECTION AGAINST DENTAL CARIES, according to claims 3 and 4, where the proposed toothpaste has a composition characterized by comprising abrasive components, between 4 and 50% w / p, among them calcium carbonate, calcium pyrophosphate, silica gel; humectant components, ranging from 20 to 65% w / w, which may be sorbitol, glycerin, polyethylene glycol, propylene glycol or mixtures thereof; binding components, between 0.5 and 7% w / w, which may be carboxymethyl cellulose, xanthan gum, thickening silica or mixture thereof; surfactant components, between 1 and 6% w / w, which may be betaine, cocoamidopropylbetaine, sodium lauryl sulfate or mixture thereof; preserving agents, between 0.01 and 0.5% w / w, which may be methylparaben, propylparaben, sodium benzoate, methyl hydroxybenzoate or mixture thereof; fluoridated agents, between 500 to 5000 ppm, Petition 870170027588, of 04/26/2017, p. 9/31 2/2 may be sodium monofluorophosphate, titanium tetrafluoride, stannous fluoride, amine fluoride, sodium fluoride or a mixture thereof; anti-plaque agents, which may be chlorhexidine, cetylpyridine chloride, triclosan and; CaneCPI-5 (26), between 0.05 and 3.00% w / w. 6) SUGAR CANE RECOMBINANT CYSTATIN FOR REDUCING EROSIVE DENTAL WEAR AND PROTECTION AGAINST DENTAL CARIES, according to claims 3 and 4, where the proposed dental gel has a composition characterized by comprising hydroxycellulose as an adjuvant component; as a binding agent glycerin; propylene glycol as a wetting component; in addition to methylparaben and imidazolidinyl urea as preservatives; deionized water, in a maximum amount of 7.5% and CaneCPI-5 (26) between 0.05 and 3.00% pp. 7) SUGARCANE RECOMBINANT CYSTATIN FOR REDUCING EROSIVE DENTAL WEAR AND PROTECTION AGAINST DENTAL CARIES, according to claims 3 and 4, where the proposed mouthwash solution contains a composition characterized by comprising deionized, flavoring, methylparaben and imidazolidininininilinin water urea as preservatives and CaneCPI-5 (26) in a concentration between 0.05 and 3.00% pp. Petition 870170027588, of 04/26/2017, p. 32/941