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.
Bezerra
Walderly Melgaço
WM
Departamento de Biologia, Centro de Ciências, Universidade Federal do Ceará, 60455-970 Fortaleza, CE, Brazil.
Carvalho
Cristina Paiva da Silveira
CP
Moreira
Renato de Azevedo
Rde A
Grangeiro
Thalles Barbosa
TB
D015153:Blotting, Western; D032449:Canavalia / Q000737:chemistry*; D018506:Gene Expression Regulation, Plant / Q000235:genetics*; D005822:Genetic Vectors; D008957:Models, Genetic; D010843:Pichia / Q000378:metabolism*; D037121:Plant Lectins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction; D019154:Protein Splicing / Q000235:genetics*
D037121:Plant Lectins; C039268:lectin, Canavalia