title: High-level expression of Falcipain-2 in Escherichia coli by codon optimization and auto-induction.
abstract: 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 1mM isopropyl-1-thio-β-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.
authors.affiliation: Centro de Estudio de Proteínas, Facultad de Biología, Universidad de la Habana, Cuba. esalas@fbio.uh.cu
authors.forename: Emir Salas
authors.initials: ES
authors.lastname: Sarduy
authors.forename: Aymara Cabrera
authors.initials: AC
authors.lastname: Muñoz
authors.forename: Sebastián Alejandro
authors.initials: SA
authors.lastname: Trejo
authors.forename: María
authors.initials: M
authors.lastname: de los A Chavéz Planes
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D003062:Codon; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D005813:Genes, Synthetic; D057166:High-Throughput Screening Assays; D006863:Hydrogen-Ion Concentration; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000201:enzymology / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012995:Solubility
chemical_list: D003062:Codon; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D003546:Cysteine Endopeptidases; C416224:falcipain 2