Cloning and heterologous expression of hyaluronidase and/or novel toxins obtained from the transcriptome of Tityus serrulatus' venom gland

• Grant number: 11/12317-3
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: March 01, 2012
• End date: November 30, 2015
• Field of knowledge: Health Sciences - Pharmacy - Toxicological Analysis
• Principal Investigator: Eliane Candiani Arantes Braga
• Grantee: Fernanda Gobbi Amorim
• Host Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated scholarship(s): 13/26083-0 - Analysis of post-translational modifications of native and recombinant toxins from Tityus serrulatus venom by mass spectrometry, BE.EP.DR

Abstract

Currently, the interest in the study of chemical and functional characteristics of toxins isolated from animal venoms is not restricted to its relevance in envenoming, but also due to their potential use as drugs in the treatment of several diseases. With the advent of "omics" techniques, such as transcriptome, it was possible not only to characterize these toxins, but also to identify new components with potential biotechnological applications. However, despite the diverse physiological actions and pharmacological potential of animal toxins, there is a large gap between the number of proteins already isolated and characterized and those that are effectively applied as drugs, which can be explained by limitations in obtainment of sufficient toxin for detailed structural and functional characterization. Thus, the heterologous expression of proteins is an viable alternative for obtaining these molecules, both in a laboratory scale, for the study of their functional and enzymatic activities, and in industrial scale, aimed at the large scale production of pharmaceuticals. Facing this challenge, this project has two main objectives, namely: the construction of a cDNA library from the venom gland of Tityus serrulatus to identify new toxins with biotechnological potential, and the cloning and expression in heterologous system of hyaluronidase and/or new toxins obtained from the transcriptome, and also to characterize them structurally and functionally to validate the heterologous expression process. Therefore, molecular biology techniques will be applied for the preparation of venom gland transcriptome, and the interesting sequences will be cloned into a vector for heterologous expression. To validate the expression, native proteins obtained from the venom, such as hyaluronidase, will be purified by chromatographic procedures and subjected to structural characterization such as mass spectrometry, N-terminal sequencing, electrophoresis, among others. In addition, native and recombinant proteins will be characterized the biochemical (enzyme kinetics, determination of isoelectric point and activity on different substrates) and functional (in vivo assays) parameters. Therefore, in this project we will validate the process of heterologous expression which may enable us to produce novel toxins bearing high biotechnological potential and others which are usually difficult to purify from venom. (AU)

Scientific publications

(10)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

AMORIM, FERNANDA GOBBI; BOLDRINI-FRANCA, JOHARA; FIGUEIREDO BORDON, KARLA DE CASTRO; CARDOSO, IARA AIME; DE PAUW, EDWIN; QUINTON, LOUC; KASHIMA, SIMONE; ARANTES, ELIANE CANDIANI. Heterologous expression of rTsHyal-1: the first recombinant hyaluronidase of scorpion venom produced in Pichia pastoris system. Applied Microbiology and Biotechnology, v. 102, n. 7, p. 3145-3158, APR 2018. (11/12317-3, 13/26083-0, 12/14996-8)

AMORIM, FERNANDA GOBBI; CORDEIRO, FRANCIELLE ALMEIDA; PINHEIRO-JUNIOR, ERNESTO LOPES; BOLDRINI-FRANCA, JOHARA; ARANTES, ELIANE CANDIANI. Microbial production of toxins from the scorpion venom: properties and applications. Applied Microbiology and Biotechnology, v. 102, n. 15, p. 6319-6331, AUG 2018. (16/04761-4, 14/16182-3, 11/12317-3, 12/14996-8)

PUCCA, MANUELA BERTO; CERNI, FELIPE AUGUSTO; PINHEIRO-JUNIOR, ERNESTO LOPES; ZOCCAL, KARINA FURLANI; FIGUEIREDO BORDON, KARLA DE CASTRO; AMORIM, FERNANDA GOBBI; PEIGNEUR, STEVE; VRIENS, KIM; THEVISSEN, KARIN; ANGELO CAMMUE, BRUNO PHILIPPE; et al. Non-disulfide-bridged peptides from Tityus serrulatus venom: Evidence for proline-free ACE-inhibitors. Peptides, v. 82, p. 44-51, AUG 2016. (13/21342-7, 14/03332-7, 12/13590-8, 13/06380-0, 14/07125-6, 13/21329-0, 11/12317-3)

BORDON, KARLA C. F.; WIEZEL, GISELE A.; AMORIM, FERNANDA G.; ARANTES, ELIANE C.. Arthropod venom Hyaluronidases: biochemical properties and potential applications in medicine and biotechnology. Journal of Venomous Animals and Toxins including Tropical Diseases, v. 21, OCT 22 2015. (11/12317-3, 14/06170-8, 11/23236-4)

CORDEIRO, FRANCIELLE A.; AMORIM, FERNANDA G.; ANJOLETTE, FERNANDO A. P.; ARANTES, ELIANE C.. Arachnids of medical importance in Brazil: main active compounds present in scorpion and spider venoms and tick saliva. Journal of Venomous Animals and Toxins including Tropical Diseases, v. 21, AUG 13 2015. (11/12317-3)

BOLDRINI-FRANCA, JOHARA; COLOGNA, CAMILA TAKENO; PUCCA, MANUELA BERTO; FIGUEIREDO BORDON, KARLA DE CASTRO; AMORIM, FERNANDO GOBBI; PINO ANJOLETTE, FERNANDO ANTONIO; CORDEIRO, FRANCIELLE ALMEIDA; WIEZEL, GISELE ADRIANO; CERNI, FELIPE AUGUSTO; PINHEIRO-JUNIOR, ERNESTO LOPES; et al. Minor snake venom proteins: Structure, function and potential applications. BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, v. 1861, n. 4, p. 824-838, APR 2017. (14/06170-8, 11/12317-3, 11/23236-4, 15/16714-8, 12/13590-8, 13/26619-7, 14/15644-3, 12/12954-6, 13/26200-6)

PUCCA, MANUELA BERTO; AMORIM, FERNANDA GOBBI; CERNI, FELIPE ATIGUSTO; FIGUEIREDO BORDON, KARLA DE CASTRO; CARDOSO, IARA AIME; PINO ANJOLETTE, FERNANDO ANTONIO; ARANTES, ELIANE CANDIANI. Influence of post-starvation extraction time and prey-specific diet in Tityus serrulatus scorpion venom composition and hyaluronidase activity. Toxicon, v. 90, p. 326-336, NOV 2014. (12/12954-6, 11/12317-3, 12/13590-8)

CERNI, FELIPE AUGUSTO; PUCCA, MANUELA BERTO; AMORIM, FERNANDA GOBBI; FIGUEIREDO BORDON, KARLA DE CASTRO; ECHTERBILLE, JULIEN; QUINTON, LOIC; DE PAUW, EDWIN; PEIGNEUR, STEVE; TYTGAT, JAN; ARANTES, ELIANE CANDIANI. Isolation and characterization of Ts19 Fragment II, a new long-chain potassium channel toxin from Tityus serrulatus venom. Peptides, v. 80, p. 9-17, JUN 2016. (13/21342-7, 12/13590-8, 13/21329-0, 13/26083-0, 11/12317-3)

PUCCA, MANUELA BERTO; CEMI, FELIPE AUGUSTO; PINHEIRO JUNIOR, ERNESTO LOPES; FIGUEIREDO BORDON, KARLA DE CASTRO; AMORIM, FEMANDA GOBBI; CORDEIRO, FRANCIELLE ALMEIDA; LONGHIM, HELOISA TAVONI; CREMONEZ, CAROLINE MARRONI; OLIVEIRA, GUILHERME HONDA; ARANTES, ELIANE CANDIANI. Tityus serrulatus venom - A lethal cocktail. Toxicon, v. 108, p. 272-284, DEC 15 2015. (12/12954-6, 11/12317-3, 14/07824-1, 12/13590-8)

CORREIA, LUCAS IAN VELOSO; DE VASCONCELOS AZEVEDO, FERNANDA VAN PETTEN; AMORIM, FERNANDA GOBBI; CIRILO GIMENES, SARAH NATALIE; POLLONI, LORENA; PEREIRA ZOIA, MARIANA ALVES; COSTA, MONICA SOARES; RODRIGUES, JESSICA PEIXOTO; GERALDO YONEYAMA, KELLY A.; SANTOS, JEAN CARLOS; et al. Shedding Lights on Crude Venom from Solitary Foraging Predatory Ant Ectatomma opaciventre: Initial Toxinological Investigation. TOXINS, v. 14, n. 1, p. 22-pg., 2022-01-01. (11/12317-3)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

AMORIM, Fernanda Gobbi. Cloning and heterologous expression of hyaluronidase and/or novel toxins obtained from the transcriptome of Tityus serrulatus\' venom gland. 2015. Doctoral Thesis - Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC) Ribeirão Preto.