Cloning and heterologous expression of the alpha-amylase inhibitor alpha- AI1 in Kluyveromyces lactis.

Brain-Isasi, Stephanie; Álvarez-Lueje, Alejandro; Corsini, Gino

Keywords: protein expression, Kluyveromyces lactis, alpha-amylase inhibitor, bean seed


Proteinaceous inhibitors of the enzyme α -amylase are widespread in plants. The seeds of several varieties of the common bean (Phaseolus vulgaris, L.) contain a plant defense glycoprotein called α -AI1, also known as phaseolamin, that inhibits the α -amylases of mammals and insects. Due to the above, this protein has a high therapeutic potential. Against this background, in this work we address the cloning and expression of α -AI1 in a Kluyveromyces lactis heterologous system. The α -AI1 codifying gene is encoded by a single locus in the genome of several varieties of P. vulgaris, so the α-AI1 gene was amplified by PCR from P. vulgaris cv. Hallado genomic DNA. This amplicon was ligated into a TOPO T/A based cloning plasmid for further transformation of Escherichia coli cell with it. The fragment with the α -AI1 codifying gene was released with the enzymes XhoI and EcoRI for the later ligation into the K. lactis expression vector pKLAC2. K. lactis positive clones for the α -AI1 gene presented an inhibitory activity against porcine pancreatic α -amylase. Acknowledgements: Conicyt Grant for Ph.D. Studies in Chile. Conicyt Grant for Ph.D. Thesis Development AT-24100168

Más información

Fecha de publicación: 2011
Año de Inicio/Término: 27-30 Septiembre, 2011
Idioma: Español