Use Of Recombinant Proteins Research Articles

Recombinant VP1 protein as a potential marker for the diagnosis of acute hepatitis A virus infection

Since hepatitis A virus (HAV) production is time-consuming and expensive, the use of recombinant proteins may represent an alternative source of antigens for diagnostic purposes. The present study aimed to express, purify and evaluate the potential of recombinant VP1 protein (rVP1) as a marker for the diagnosis of acute HAV infection. The rVP1 was expressed and purified successfully from Escherichia coli. The purified rVP1 was used to establish an in-house enzyme-linked immunosorbent assay (ELISA-rVP1) for detection of IgM antibodies in sera from HAV-positive patients. For a cut-off point of 0.351, the sensitivity and specificity of ELISA-rVP1 were 100.0% and 95.0%, respectively. These results indicate that rVP1 may be a useful antigen for detection of IgM antibodies against HAV.

Pneumococcal Predictive Proteins Selected by Microbial Genomic Approach Are Serotype Cross-Reactive and Bind to Host Extracellular Matrix Proteins.

Streptococcus pneumoniae is a colonizer of the human nasopharynx, which accounts for most of the community-acquired pneumonia cases and can cause non-invasive and invasive diseases. Current available vaccines are serotype-specific and the use of recombinant proteins associated with virulence is an alternative to compose vaccines and to overcome these problems. In a previous work, we describe the identification of proteins in S. pneumoniae by reverse vaccinology and the genetic diversity of these proteins in clinical isolates. It was possible to purify a half of 20 selected proteins in soluble form. The expression of these proteins on the pneumococcal cells surface was confirmed by flow cytometry. We demonstrated that some of these proteins were able to bind to extracellular matrix proteins and were recognized by sera from patients with pneumococcal meningitis infection caused by several pneumococcal serotypes. In this context, our results suggest that these proteins may play a role in pneumococcal pathogenesis and might be considered as potential vaccine candidates.

Identification, production and assessment of two Toxoplasma gondii recombinant proteins for use in a Toxoplasma IgG avidity assay

The IgG avidity assay is an important tool in the management of suspected toxoplasmosis in pregnant women. This study aimed to produce new Toxoplasma gondii recombinant proteins and to assess their usefulness in an IgG avidity assay. Toxoplasma positive and negative serum samples were used, the former were categorized into low (LGA) and high (HGA) IgG avidity samples. Immunoblots were performed on 30 T. gondii cDNA clones to determine the reactivity and IgG avidity to the expressed proteins. Two of the clones were found to have diagnostic potential and were analyzed further; AG12b encoded T. gondii apical complex lysine methyltransferase (AKMT) protein and AG18 encoded T. gondii forkhead-associated (FHA) domain-containing protein. The His-tagged recombinant proteins, rAG12b and rAG18, were expressed and tested with LGA and HGA samples using an IgG avidity western blot and ELISA. With the IgG avidity western blot, rAG12b identified 86.4% of LGA and 90.9% of HGA samples, whereas rAG18 identified 81.8% of both LGA and HGA samples. With the IgG avidity ELISA, rAG12b identified 86.4% of both LGA and HGA samples, whereas rAG18 identified 77.3% of LGA and 86.4% of HGA serum samples. This study showed that the recombinant antigens were able to differentiate low avidity and high avidity serum samples, suggesting that they are potential candidates for use in the Toxoplasma IgG avidity assay.

Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system

The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS.

Calcyclin Binding Protein/Siah-1 Interacting Protein Is a Hsp90 Binding Chaperone.

The Hsp90 chaperone activity is tightly regulated by interaction with many co-chaperones. Since CacyBP/SIP shares some sequence homology with a known Hsp90 co-chaperone, Sgt1, in this work we performed a set of experiments in order to verify whether CacyBP/SIP can interact with Hsp90. By applying the immunoprecipitation assay we have found that CacyBP/SIP binds to Hsp90 and that the middle (M) domain of Hsp90 is responsible for this binding. Furthermore, the proximity ligation assay (PLA) performed on HEp-2 cells has shown that the CacyBP/SIP-Hsp90 complexes are mainly localized in the cytoplasm of these cells. Using purified proteins and applying an ELISA we have shown that Hsp90 interacts directly with CacyBP/SIP and that the latter protein does not compete with Sgt1 for the binding to Hsp90. Moreover, inhibitors of Hsp90 do not perturb CacyBP/SIP-Hsp90 binding. Luciferase renaturation assay and citrate synthase aggregation assay with the use of recombinant proteins have revealed that CacyBP/SIP exhibits chaperone properties. Also, CacyBP/SIP-3xFLAG expression in HEp-2 cells results in the appearance of more basic Hsp90 forms in 2D electrophoresis, which may indicate that CacyBP/SIP dephosphorylates Hsp90. Altogether, the obtained results suggest that CacyBP/SIP is involved in regulation of the Hsp90 chaperone machinery.

A Lipid Based Antigen Delivery System Efficiently Facilitates MHC Class-I Antigen Presentation in Dendritic Cells to Stimulate CD8+ T Cells

The most effective strategy for protection against intracellular infections such as Leishmania is vaccination with live parasites. Use of recombinant proteins avoids the risks associated with live vaccines. However, due to low immunogenicity, they fail to trigger T cell responses particularly of CD8+ cells requisite for persistent immunity. Previously we showed the importance of protein entrapment in cationic liposomes and MPL as adjuvant for elicitation of CD4+ and CD8+ T cell responses for long-term protection. In this study we investigated the role of cationic liposomes on maturation and antigen presentation capacity of dendritic cells (DCs). We observed that cationic liposomes were taken up very efficiently by DCs and transported to different cellular sites. DCs activated with liposomal rgp63 led to efficient presentation of antigen to specific CD4+ and CD8+ T cells. Furthermore, lymphoid CD8+ T cells from liposomal rgp63 immunized mice demonstrated better proliferative ability when co-cultured ex vivo with stimulated DCs. Addition of MPL to vaccine enhanced the antigen presentation by DCs and induced more efficient antigen specific CD8+ T cell responses when compared to free and liposomal antigen. These liposomal formulations presented to CD8+ T cells through TAP-dependent MHC-I pathway offer new possibilities for a safe subunit vaccine.

Insect Larvae: A New Platform to Produce Commercial Recombinant Proteins.

In Biotechnology, the expression of recombinant proteins is a constantly growing field and different hosts are used for this purpose. Some valuable proteins cannot be produced using traditional systems. Insects from the order Lepidoptera infected with recombinant baculovirus have appeared as a good choice to express high levels of proteins, especially those with post-translational modifications. Lepidopteran insects, which are extensively distributed in the world, can be used as small protein factories, the new biofactories. Species like Bombyx mori (silkworm) have been analyzed in Asian countries to produce a great number of recombinant proteins for use in basic and applied science and industry. Many proteins expressed in this larva have been commercialized. Several recombinant proteins produced in silkworms have already been commercialized. On the other hand, species like Spodoptera frugiperda, Heliothis virescens, Rachiplusia nu, Helicoverpa zea and Trichoplusia ni are widely distributed in both the occidental world and Europe. The expression of recombinant proteins in larvae has the advantage of its low cost in comparison with insect cell cultures. A wide variety of recombinant proteins, including enzymes, hormones and vaccines, have been efficiently expressed with intact biological activity. The expression of pharmaceutically proteins, using insect larvae or cocoons, has become very attractive. This review describes the use of insect larvae as an alternative to produce commercial recombinant proteins.

Tuning Mesenchymal Stem Cell Response onto Titanium–Niobium–Hafnium Alloy by Recombinant Fibronectin Fragments

Since metallic biomaterials used for bone replacement possess low bioactivity, the use of cell adhesive moieties is a common strategy to improve cellular response onto these surfaces. In recent years, the use of recombinant proteins has emerged as an alternative to native proteins and short peptides owing to the fact that they retain the biological potency of native proteins, while improving their stability. In the present study, we investigated the biological effect of two different recombinant fragments of fibronectin, spanning the 8-10th and 12-14th type III repeats, covalently attached to a new TiNbHf alloy using APTES silanization. The fragments were studied separately and mixed at different concentrations and compared to a linear RGD, a cyclic RGD and the full-length fibronectin protein. Cell culture studies using rat mesenchymal stem cells demonstrated that low to medium concentrations (30% and 50%) of type III 8-10th fragment mixed with type III 12-14th fragment stimulated cell spreading and proliferation compared to RGD peptides and the fragments separately. On the other hand, type III 12-14th fragment alone or mixed at low volume percentages ≤50% with type III 8-10th fragment increased alkaline phosphatase levels compared to the other molecules. These results are significant for the understanding of the role of fibronectin recombinant fragments in cell responses and thus to design bioactive coatings for biomedical applications.

SCREENING OF CHLOROPLAST PROMOTERS FOR HEVEA BRASILIENSIS CHLOROPLAST TRANSFORMATION

In recent years, the growth in the use of recombinant proteins has grown tremendously. With the aid of the advances in DNA recombinant biotechnology, molecular farming in plants has been applied to meet this increasing demand where plants have emerged as one of the most promising general production platforms for recombinant proteins. Hevea brasiliensis is one of the main commodities in Malaysia and widely cultivated species for commercial production of latex. This important plant has been used to express recombinant proteins such as a single-chain variable fragment (scFv) antibody against the coat protein of Streptococcus gordonii (an oral dental bacterium), human serum albumin and human atrial natriuretic. The genes that encodes for the recombinant proteins were targeted into the nucleus genome of Hevea but the proteins were expressed in low concentration. Generating transgenic plant using chloroplast transformation offers many advantages in comparison to nuclear transformation and many researches have been made to apply this strategy to enhance agronomic traits or produce recombinant protein in several plant species. Since chloroplast is highly polyploidy, it allows high-level foreign protein expression. Given the generally very high foreign protein accumulation rates that can be achieved in transgenic chloroplasts, the aim of this study is to screen a number of chosen endogenous Hevea chloroplast promoters to drive the expression of the reporter gene, uidA for Hevea specific chloroplast transformation vector. Three promoters were chosen for this experiment which are; rbcL, psbA and rrn16 promoters. The putative regions of these promoters were derived from the chloroplast genome sequence of Hevea. Analyses of the three putative promoter regions using multiple sequence alignment with comparable regions from other plant species show significant sequence homology. Further analyses of the putative regions using in-vitro transcription are planned for future study. It is hoped that with the development of an optimized expression vector will allow high expression of valuable recombinant protein in the chloroplast of Hevea.

The structure of the Guanine Nucleotide Exchange Factor Rlf in complex with the small G-protein Ral identifies conformational intermediates of the exchange reaction and the basis for the selectivity.

CDC25 homology domain (CDC25-HD) containing Guanine Nucleotide Exchange Factors (GEFs) initiate signalling by small G-proteins of the Ras-family. Each GEF acts on a small subset of the G-proteins only, thus providing signalling selectivity. Rlf is a GEF with selectivity for the G-proteins RalA and RalB. Here the crystal structure of Rlf in complex with Ral is determined. The Rlf·Ral complex crystallised into two different crystal forms, which represent different steps of the exchange reaction. Thereby general insight in the CDC25-HD catalysed nucleotide exchange is obtained. In addition, the basis for the selectivity of the interaction is investigated. The exchange activity is monitored by the use of recombinant proteins. Selectivity determinants in the binding interface are identified and confirmed by a mutational study.

Silicon Crystals Formation Using Silicatein-Like Cathepsin of Marine Sponge <I>Latrunculia oparinae</I>

The cDNA fragment encoding the catalytic domain of the new silicatein-like cathepsin enzyme LoCath was expressed in a strain Top10 of Escherichia coli, extracted and purified via nickel-affinity chromatography. Recombinant enzyme performed silica-polymerizing activity when mixed with water-soluble silica precursor-tetrakis-(2-hydroxyethyl)-orthosilicate. Scanning electron microscopy revealed hexagonal, octahedral and β-tridimit crystals. Energy dispersion fluorescence X-ray spectrometry analysis showed that all these crystals consist of pure silicon oxide. It is the first report about the ability of marine sponge's cathepsin to polymerize silicon, as well as about the structure and composition of the silicon oxide crystal formed by recombinant cathepsin. Further study of the catalytic activity of silicatein and cathepsin will help to understand the biosilification processes in vivo, and will create basis for biotechnological use of recombinant proteins for silicon polymerization.

Different expression systems for production of HCV structural proteins

Hepatitis C virus (HCV) is an important agent causing chronic liver infection, which often leads to liver cirrhosis and lethal hepatocellular carcinoma (HCC). At present, there is no effective HCV vaccine for prevention of hepatic disease and the standard treatment is neither economical nor fully effective in all the patients. However, vaccination based on structural and nonstructural proteins of HCV has attracted a special interest. Different heterologous systems have been used to generate the recombinant HCV core, E1, and E2 proteins including Escherichia coli , yeast, insects and mammalian cells. Further studies showed that the amounts of HCV recombinant proteins in E. coli are more suitable and un-expensive compared to other systems. It should be considered that this system is not efficient for generation of the glycosylated proteins. Thus, the structure of proteins is an important agent of selection for expression systems. The selection of expression systems will be critical for the use of recombinant proteins as an immunogen. In this mini-review, we briefly describe different expression systems for generation of the HCV recombinant structural proteins applied in vaccine design.

Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast

There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.

Regulation of Plasmodium falciparum Origin Recognition Complex subunit 1 (PfORC1) function through phosphorylation mediated by CDK-like kinase PK5.

Plasmodium falciparum Origin Recognition Complex subunit 1 (PfORC1) has been implicated in DNA replication and var gene regulation. While the C-terminus is involved in DNA replication, the specific role of N-terminus has been suggested in var gene regulation in a Sir2-dependent manner. PfORC1 is localized at the nuclear periphery, where the clustering of chromosomal ends at the early stage of parasite development may be crucial for the regulation of subtelomeric var gene expression. Upon disassembly of telomeric clusters at later stages of parasite development, ORC1 is distributed in the nucleus and parasite cytoplasm where it may be required for its other cellular functions including DNA replication. The level of ORC1 decreases dramatically at the late schizont stage. The mechanisms that mediate regulation of PfORC1 function are largely unknown. Here we show, by the use of recombinant proteins and of transgenic parasites expressing wild type or mutant forms of ORC1, that phosphorylation of the PfORC1-N terminal domain by the cyclin-dependent kinase (CDK) PfPK5 abolishes DNA-binding activity and leads to changes in subcellular localization and proteasome-mediated degradation of the protein in schizonts. These results reveal that PfORC1 phosphorylation by a CDK is central to the regulation of important biological functions like DNA replication and var gene silencing.

An Improved Nonradioactive Screening Method Identifies Genistein and Xanthohumol as Potent Inhibitors of Iodothyronine Deiodinases.

Deiodinases (DIO1, 2, and 3) are key enzymes in thyroid hormone (TH) activation and inactivation with impact on energy metabolism, development, cell differentiation, and a number of other physiological processes. The three DIO isoenzymes thus constitute sensitive rate-limiting components within the TH axis, prone to dysregulation by endocrine disruptive compounds or disease state. In animal models and cell culture experiments, they serve as readout for local TH status and disarrangement of the hormonal axis. Furthermore, some human diseases are characterized by apparent deiodinase dysregulation (e.g., the low triiodothyronine syndrome in critical illness). Consequently, these enzymes are targets of interest for the development of pharmacological compounds with modulatory activities. Until now, the portfolio of inhibitors for these enzymes is limited. In the clinics, the DIO1-specific inhibitor propylthiouracil is in use for treatment of severe hyperthyroidism. Other well-known inhibitors (e.g., iopanoic acid or aurothioglucose) are nonselective and block all three isoenzymes. Furthermore, DIO3 was shown to be a potential oncogenic gene, which is strongly expressed in some tumors and might, in consequence, protect tumor tissue form differentiation by TH. With respect to its role in tumorigenesis, specific inhibitors of DIO3 as a potential target for anticancer drugs would be highly desirable. To this end, a flexible and convenient assay for high-throughput screening is needed. We recently described a nonradioactive screening assay, utilizing the classic Sandell-Kolthoff reaction as readout for iodide release from the substrate molecules. While we used murine liver as enzyme source, the assay was limited to murine DIO1 activity testing. Here, we describe the use of recombinant proteins as enzyme sources within the assay, expanding its suitability from murine Dio1 to human DIO1, DIO2, and DIO3. As proof-of-concept, deiodination reactions catalyzed by these recombinant enzymes were monitored with various nonradioactive substrates and confirmed by liquid chromatography-tandem mass spectrometry. The contrast agent and known DIO inhibitor iopanoic acid was characterized as readily accepted substrate by DIO2 and Dio3. In a screening approach using established endocrine disrupting compounds, the natural food ingredient genistein was identified as a further DIO1-specific inhibitor, while xanthohumol turned out to potently block the activity of all three isoenzymes. A rapid nonradioactive screening method based on the Sandell-Kolthoff reaction is suitable for identification of environmental, nutritive and pharmacological compounds modulating activities of human deiodinase enzymes.

Heterologously expressed Aspergillus aculeatus β-glucosidase in Saccharomyces cerevisiae is a cost-effective alternative to commercial supplementation of β-glucosidase in industrial ethanol production using Trichoderma reesei cellulases

Trichoderma reesei is a filamentous organism that secretes enzymes capable of degrading cellulose to cellobiose. The culture supernatant of T.reesei, however, lacks sufficient activity to convert cellobiose to glucose using β-glucosidase (BGL1). In this study, we identified a BGL (Cel3B) from T.reesei (TrCel3B) and compared it with the active β-glucosidases from Aspergillus aculeatus (AaBGL1). AaBGL1 showed higher stability and conversion of sugars to ethanol compared to TrCel3B, and therefore we chose to express this recombinant protein for use in fermentation processes. We expressed the recombinant protein in the yeast Saccharomyces cerevisiae, combined it with the superb T.reesei cellulase machinery and used the combination in a simultaneous saccharification and fermentation (SSF) process, with the hope that the recombinant would supplement the BGL activity. As the sugars were processed, the yeast immediately converted them to ethanol, thereby eliminating the problem posed by end product inhibition. Recombinant AaBGL1 activity was compared with Novozyme 188, a commercially available supplement for BGL activity. Our results show that the recombinant protein is as effective as the commercial supplement and can process sugars with equal efficiency. Expression of AaBGL1 in S.cerevisiae increased ethanol production effectively. Thus, heterologous expression of AaBGL1 in S.cerevisiae is a cost-effective and efficient process for the bioconversion of ethanol from lignocellulosic biomass.

Fine mapping and characterization of the L-polymerase-binding domain of the respiratory syncytial virus phosphoprotein.

The minimum requirement for an active RNA-dependent RNA polymerase of respiratory syncytial virus (RSV) is a complex made of two viral proteins, the polymerase large protein (L) and the phosphoprotein (P). Here we have investigated the domain on P that is responsible for this critical P-L interaction. By use of recombinant proteins and serial deletions, an L binding site was mapped in the C-terminal region of P, just upstream of the N-RNA binding site. The role of this molecular recognition element of about 30 amino acid residues in the L-P interaction and RNA polymerase activity was evaluated in cellula using an RSV minigenome system and site-directed mutagenesis. The results highlighted the critical role of hydrophobic residues located in this region. Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine and no good antivirals against RSV are available, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. Like all negative-strand RNA viruses, RSV codes for its own machinery to replicate and transcribe its genome. The core of this machinery is composed of two proteins, the phosphoprotein (P) and the large protein (L). Here, using recombinant proteins, we have mapped and characterized the P domain responsible for this L-P interaction and the formation of an active L-P complex. These findings extend our understanding of the mechanism of action of RSV RNA polymerase and allow us to define a new target for the development of drugs against RSV.

Production and immunological analysis of IgE reactive recombinant egg white allergens expressed in Escherichia coli

IgE-mediated allergy to chicken egg affects a large number of children and adults worldwide. The current management strategy for egg allergy is strict avoidance, however this is impractical due to the presence of eggs in a range of foods and pharmaceutical products including vaccines. Strict avoidance also poses nutritional disadvantages due to high nutritional value of eggs. Allergen specific immunotherapy is being pursued as a curative treatment, in which an allergic individual is gradually exposed to the allergen to induce tolerance. Use of recombinant proteins for immunotherapy has been beneficial due to the purity of the recombinant proteins compared to natural proteins. In this study, we produced IgE reactive recombinant egg white proteins that can be used for future immunotherapy. Using E. coli as an expression system, we successfully produced recombinant versions of Gal d 1, 2 and 3, that were IgE reactive when tested against a pool of egg allergic patients’ sera. The IgE reactivity indicates that these recombinant proteins are capable of eliciting an immune response, thus being potential candidates for immunotherapy. We have, for the first time, attempted to produce recombinant versions of all 4 major egg white allergens in E. coli, and successfully produced 3, with only Gal d 4 showing loss of IgE reactivity in the recombinant version. The results suggest that egg allergy in Australian populations may mainly be due to IgE reactivity to Gal d 3 and 4, while Gal d 1 shows higher IgE reactivity. This is the first report of a collective and comparative immunological analysis of all 4 egg white allergens. The significance of this study is the potential use of the IgE reactive recombinant egg white proteins in immunotherapy to treat egg allergic patients.

New and emerging strategies in platelet-rich plasma application in musculoskeletal regenerative procedures: general overview on still open questions and outlook.

Despite its pervasive use, the clinical efficacy of platelet-rich plasma (PRP) therapy and the different mechanisms of action have yet to be established. This overview of the literature is focused on the role of PRP in bone, tendon, cartilage, and ligament tissue regeneration considering basic science literature deriving from in vitro and in vivo studies. Although this work provides evidence that numerous preclinical studies published within the last 10 years showed promising results concerning the application of PRP, many key questions remain unanswered and controversial results have arisen. Additional preclinical studies are needed to define the dosing, timing, and frequency of PRP injections, different techniques for delivery and location of delivery, optimal physiologic conditions for injections, and the concomitant use of recombinant proteins, cytokines, additional growth factors, biological scaffolds, and stems cells to develop optimal treatment protocols that can effectively treat various musculoskeletal conditions.

Evaluation of the use of recombinant proteins of Mycobacterium bovis as antigens in intradermal tests for diagnosis of bovine tuberculosis

La prueba itradermica de la piel con derivados de proteina (PPD) purificada a partir de Mycobacterium bovis se ha utilizado para el diagnostico de la tuberculosis bovina. Sin embargo, debido a la especificidad suboptima de esta mezcla de proteinas, la mejora de las pruebas basadas en antigenos especificos definidos son necesarias. En el presente estudio, las proteinas recombinantes de M. bovis se evaluaron como antigenos en la prueba cutanea. Entre estas, EsxI, Mb0143, PE5 y PE13 son antigenos probados por primera vez en el ganado. Bovinos sensibilizados y no sensibilizados con la cepa AN5 inactivada de M. bovis fueron inyectados simultaneamente con cada proteina recombinante, un coctel con todas las proteinas recombinantes, PPD de M. bovis, PPD de M. avium y salina. Solo las proteinas EsxI, Mb0143 y PE5 fueron capaces de diferenciar ganado sensibilizado de lo no sensibilizado cuando se uso 320 irg de proteina y la lectura realizada 24 horas despues de la inyeccion. EsxI y PE5 exhibieron un nivel de sensibilidad de 83,33% y una especificidad de 100% y 80%, respectivamente. Los resultados del presente estudio sugieren que las proteinas recombinantes tienen el potencial de ser evaluadas como antigenos en las pruebas cutaneas en el ganado