Establishment of a heterologous system for the expression of Canavalia brasiliensis lectin: a model for the study of protein splicing

During its biosynthesis in developing Canavalia brasiliensis seeds, the lectin ConBr undergoes a form of protein splicing in which the order of the N- and C-domains of the protein is reversed. To investigate whether these events can occur in other eukaryotic organisms, an expression system based on Pichia pastoris cells was established. A DNA fragment encoding prepro-ConBr was cloned into the vector pPICZB, and the recombinant plasmid was transformed in P. pastoris strain GS115. Ten clones were screened for effective recombinant protein production. Based on Western blot analysis of the two clones with the highest level of protein expression: 1) diffuse high-molecular mass immunoreactive bands were produced as early as 24 h after induction; 2) a single-, high-molecular mass protein was secreted into the medium, and 3) a significant fraction of the recombinant polypeptides that cross-reacted with anti-ConBr antibodies comprised a band of approximately 34.5 kDa. Diffuse protein bands with high molecular masses are attributed to hyperglycosylation at the single potential N-glycosylation site located in the linker peptide of prepro-ConBr. In contrast, native ConBr is made up of three polypeptides, the intact alpha chain (aa 1-237) and the fragments beta (aa 1-118) and gamma (aa 119-237), which have apparent molecular masses of 30, 16 and 12 kDa, respectively. Apparently, the yeast P. pastoris is not able to carry out all the complex post-translational proteolytic processing necessary for the biosynthesis of ConBr.

Efecto de la extrusión sobre la actividad de factores antinutricionales y digestibilidad in vitro de proteínas y almidón en harinas de Canavalia ensiformis / Effect of extrusion on the activity of anti-nutritional factors and in vitro digestibility of protein and starch in flours of Canavalia ensiformis

The effect of the extrusion (155 degrees C, 20 per cent moisture, screw speed 75 rpm, feed speed 205 g min-1) on antinutritional factors of Canavalia ensiformis was studied. In vitro protein and starch digestibilities were assessed. The extrusion not affect protein content (23 per cent) in the flours, but significantly (P < 0.01) decrease moisture content. The protein digestibility values were improved from 57.5 to 89.5 per cent, these values were lower than casein (98.19 per cent). The digestibility of starch values were improved from 37.7 to 53 per cent. The protease inhibitors activities (trypsin and chymotrypsin) and alpha-amylase inhibitor activity were reduced by 95 per cent. The haemagglutinating activity was eliminated as result of the high temperature employed during the extrusion process. The canavanine content in the flours were not affect by the treatment of extrusion.