Establishment of a heterologous system for the expression of Canavalia brasiliensis lectin: a model for the study of protein splicing
Genet. mol. res. (Online)
;
5(1): 216-223, Mar. 31, 2006. graf, ilus
Artículo
en Inglés
| LILACS
| ID: lil-449131
ABSTRACT
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.
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Índice:
LILACS (Américas)
Asunto principal:
Pichia
/
Regulación de la Expresión Génica de las Plantas
/
Empalme de Proteína
/
Canavalia
/
Lectinas de Plantas
/
Modelos Genéticos
País/Región como asunto:
America del Sur
/
Brasil
Idioma:
Inglés
Revista:
Genet. mol. res. (Online)
Asunto de la revista:
Biologia Molecular
/
Genética
Año:
2006
Tipo del documento:
Artículo
País de afiliación:
Brasil
Institución/País de afiliación:
Universidade Estadual do Ceará/BR
/
Universidade Federal do Ceará/BR
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