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(Referência obtida automaticamente do Web of Science, por meio da informação sobre o financiamento pela FAPESP e o número do processo correspondente, incluída na publicação pelos autores.)

Heterologous expression of rTsHyal-1: the first recombinant hyaluronidase of scorpion venom produced in Pichia pastoris system

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Autor(es):
Amorim, Fernanda Gobbi [1] ; Boldrini-Franca, Johara [1] ; Figueiredo Bordon, Karla de Castro [1] ; Cardoso, Iara Aime [1] ; De Pauw, Edwin [2] ; Quinton, Louc [2] ; Kashima, Simone [3] ; Arantes, Eliane Candiani [1]
Número total de Autores: 8
Afiliação do(s) autor(es):
[1] Univ Sao Paulo, Sch Pharmaceut Sci Ribeirao Preto, Dept Chem & Phys, Av Cafe S-N, BR-14040903 Ribeirao Preto - Brazil
[2] Univ Liege, Dept Chem, Liege - Belgium
[3] Univ Sao Paulo, Ribeirao Preto Med Sch, Reg Ctr Hemotherapy Ribeirao Preto, Sao Paulo - Brazil
Número total de Afiliações: 3
Tipo de documento: Artigo Científico
Fonte: Applied Microbiology and Biotechnology; v. 102, n. 7, p. 3145-3158, APR 2018.
Citações Web of Science: 4
Resumo

In general, hyaluronidases have a broad potential application on medicine and esthetics fields. Hyaluronidases from animal venoms cleave hyaluronan present in the extracellular matrix, acting as spreading factors of toxins into the tissues of the victim. However, the in-depth characterization of hyaluronidase from animal venoms has been neglected due to its instability and low concentration in the venom, which hamper its isolation. Thus, heterologous expression of hyaluronidase acts as a biotechnological tool in the obtainment of enough amounts of the enzyme for structural and functional studies. Therefore, this study produced a recombinant hyaluronidase from Tityus serrulatus scorpion venom, designated as rTsHyal-1, in the Pichia pastoris system. Thus, a gene for TsHyal-1 (gb|KF623285.1) was synthesized and cloned into the pPICZ alpha A vector (GenScript Corporation) for heterologous expression in P. pastoris. rTsHyal-1 was expressed in laboratorial scale in a buffered minimal medium containing methanol (BMM) for 96 h with daily addition of methanol. Expression of rTsHyal-1 resulted in a total protein yield of 0.266 mg/mL. rTsHyal-1 partially purified through cation exchange chromatography presented a specific activity of 1097 TRU/mg, against 838 TRU/mg for the final expressed material, representing a 1.31-fold purification. rTsHyal-1 has molecular mass of 49.5 kDa, and treatment with PNGase F and analysis by mass spectrometry (MALDI-TOF) indicated a potential N-glycosylation of 4.5 kDa. Additionally, de novo sequencing of rTsHyal-1, performed in MALDI-TOF and Q Exactive Orbitrap MS, resulted in 46.8% of protein sequence coverage. rTsHyal-1 presents the highest substrate specificity to hyaluronan followed by chondroitin-6-sulfate, chondroitin-4-sulfate, and dermatan sulfate and showed an optimum activity at pH 6.0 and 40 A degrees C. These results validate the biotechnological process for the heterologous expression of rTsHyal-1. This is the first recombinant hyaluronidase from scorpion venoms expressed in the P. pastoris system with preserved enzyme activity. (AU)

Processo FAPESP: 13/26083-0 - Análise de modificações pós-traducionais de toxinas nativas e recombinantes da peçonha Tityus serrulatus por espectrometria de massas
Beneficiário:Fernanda Gobbi Amorim
Linha de fomento: Bolsas no Exterior - Estágio de Pesquisa - Doutorado
Processo FAPESP: 12/14996-8 - Clonagem e expressão de toxinas animais de interesse biotecnológico
Beneficiário:Eliane Candiani Arantes Braga
Linha de fomento: Auxílio à Pesquisa - Regular
Processo FAPESP: 11/12317-3 - Clonagem e expressão heteróloga da hialuronidase e/ou novas toxinas obtidas a partir do transcriptoma da glândula da peçonha de Tityus serrulatus
Beneficiário:Fernanda Gobbi Amorim
Linha de fomento: Bolsas no Brasil - Doutorado