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Frutapin, a lectin from Artocarpus incisa (breadfruit): cloning, expression and molecular insights

DOI: 10.1042/BSR20170969 DOI Help

Authors: Felipe Domingos De Sousa (University of Fortaleza (UNIFOR); Federal University of Ceará (UFC)) , Bruno Bezerra Da Silva (State University of Ceará (UECE)) , Gilvan Pessoa Furtado (Fundação Oswaldo Cruz - Ceará) , Igor De Sa Carneiro (University of Fortaleza (UNIFOR)) , Marina Duarte Pinto Lobo (University of Fortaleza (UNIFOR)) , Yiwei Guan (University College London) , Jingxu Guo (University College London) , Alun R. Coker (University College London) , Marcos Roberto Lourenzoni (Fundação Oswaldo Cruz - Ceará) , Maria Izabel Florindo Guedes (State University of Ceará (UECE)) , James S. Owen (University College London) , David J. Abraham (University College London) , Ana Cristina De Oliveira Monteiro-moreira (University of Fortaleza (UNIFOR)) , Renato De Azevedo Moreira
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Bioscience Reports

State: Published (Approved)
Published: July 2017
Diamond Proposal Number(s): 12342 , 12342

Abstract: Artocarpus incisa (breadfruit) seeds contain three different lectins (Frutalin, Frutapin and Frutackin) with distinct carbohydrate specificities. The most abundant lectin is Frutalin, an α-D-galactose-specific carbohydrate-binding glycoprotein with antitumour properties and potential for tumour biomarker discovery as already reported. Frutapin (FTP) is the second most abundant, but proved difficult to purify with very low yields and contamination with Frutalin frustrating its characterization. Here, we report for the first time high-level production and isolation of biologically-active recombinant FTP in E. coli BL21, optimizing conditions with the best set yielding >40 mg/L culture of soluble active FTP. The minimal concentration for agglutination of red blood cells was 62.5 µg/mL of FTP, a process effectively inhibited by mannose. Apo-FTP, FTP-mannose and FTP-glucose crystals were obtained and diffracted X-rays to a resolution of 1.58 (P212121), 1.70 (P3121) and 1.60 (P3121) Å, respectively. The best solution showed four monomers per asymmetric unit. Molecular Dynamics simulation suggested FTP displays higher affinity for mannose than glucose. Cell studies revealed FTP was non-cytotoxic to cultured mouse fibroblast 3T3 cells below 0.5 mg/mL and also capable of stimulating cell migration at 50 µg/mL. In conclusion, our optimized expression system allowed high amounts of correctly-folded soluble FTP to be isolated. This recombinant bioactive lectin will now be tested in future studies for therapeutic potential; for example, in wound healing and tissue regeneration.

Subject Areas: Biology and Bio-materials

Instruments: I02-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography

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