High-level expression of Falcipain-2 in Escherichia coli by codon optimization and auto-induction

• Autores: Salas Sarduy, Emir; Cabrera Muñoz, Aymara; Trejo, Sebastián Alejandro; Chavéz Planes, María de los A.
• Año de publicación: 2012
• Idioma: inglés
• Tipo de recurso: artículo
• Materia: Bioquímica y Biología Molecular; Falcipain-2; Synthetic gene; Codon optimization; Auto-induction; Plasmodium falciparum
• Institución: Comisión de Investigaciones Científicas de la Provincia de Buenos Aires

Falcipain-2, the major cysteine hemoglobinase from the human malaria parasite Plasmodium falciparum, is critical for parasite development and is considered a promising chemotherapeutic target. In order to facilitate the high-throughput screening of Falcipain-2 inhibitors from natural sources, we developed an economic and highly-productive overexpression system in Escherichia coli using a codon-optimized proFalcipain-2 construct. Very high expression levels (35–55% of total host proteins) were observed when proFalcaipain-2 expression was induced with 1 mM isopropyl-1-thio-b-D-galactopyranoside (IPTG) in several E. coli strains, with the highest level observed for BL21(DE3). A lower expression ( 40% of total host proteins) was observed when BL21(DE3) was grown in ZYM-5052 auto-induction medium, containing 0.2% lactose as inducer. However, the culture grew to notably higher cellular density, increasing 1.5 times the overall yield of the system when compared with conventional IPTG-induction. Although several conditions were modified to achieve the expression of soluble and active Falcipain-2, the enzyme was mainly obtained in the form of insoluble aggregates. After purification and refolding, 50 mg of active enzyme were obtained per liter of culture at low cost using a regular incubator shaker, and recombinant Falcipain-2 exhibited structural and functional characteristics very similar to the natural counterpart. Due to its versatility and simplicity, this strategy can be straightforwardly adapted to other proteins from Plasmodium species or any other organism with an AT-rich genome.