Igor de Sá Carneiro

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

Artocarpus incisa (breadfruit) seeds contain three different lectins (Frutalin, Frutapin (FTP) 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. 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 Escherichia 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 they 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 (MD) simulation suggested that FTP displays higher affinity for mannose than glucose. Cell studies revealed that FTP was non-cytotoxic to cultured mouse fibroblast 3T3 cells below 0.5 mg/ml and was 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.

Effects of oocyte source, cell origin, and embryo reconstruction procedures on in vitro and in vivo embryo survival after goat cloning

The birth of cloned goats has been well documented, but the overall goat cloning efficiency by somatic cell nuclear transfer procedures is still low, which may be further intensified in extreme environments. The aim of this study was to produce cloned goats under the conditions of the Brazilian SemiArid region, in a transgenic program for the expression of human lysozyme in the milk to target childhood diarrhea and malnutrition, comparing the effects of oocyte source, cell type, and embryo reconstruction procedures on in vitro and in vivo embryo survival after cloning by micromanipulation or by handmade cloning. The use of in vitro-matured oocytes resulted in more viable embryos after cloning than in vivo-matured cytoplasts, but no differences in pregnancy rates on day 23 were seen between oocyte sources (77.5 vs. 77.8%, respectively). The presence or absence of the zona pellucida for embryo reconstruction (78.8 vs. 76.0%, respectively) did not affect pregnancy outcome after transfer. However, pregnancy rate on day 23 was higher for embryos chemically activated by a conventional than a modified protocol (88.1 vs. 50.0%), and for embryos reconstructed with mesenchymal stem cells and fetal fibroblasts (100.0 and 93.3%) than with adult fibroblasts (64.7%). Although most pregnancies were lost, the birth of a cloned female was obtained from embryos reconstructed by micromanipulation using non-transgenic control cells and in vitro-matured oocytes with intact zona pellucida, after conventional activation and transfer at the 1-cell stage.