title: A platform for high-throughput expression of recombinant human enzymes secreted by insect cells.
abstract: Functional genomics and proteomics have been fields of intense investigation, since the disclosure of the sequence of the human genome. To contribute to the assignment of a physiological role to the vast number of coding genes with unknown function, we have undertaken a program to clone, express, purify and determine the catalytic activity of those enzymes predicted to enter the secretory pathway, focusing our efforts on human peptidases. Our strategy to promote high-throughput expression and purification of recombinant proteins secreted by insect cells relies on the expression of the target enzymes with their native leader sequences and on the carboxyl-terminal fusion with a poly-histidine tag. Growth of host cells were optimized in 24-well format to achieve highly paralleled culture conditions with production yields comparable to shake flask. The purification was performed by a robotic system in 96-well format using either magnetic beads or minicolumns. In a pilot study using reference peptidases and lipases, the high-throughput approach demonstrated to support the secretion in the insect cell medium of 85% of the sample enzymes. Of them, 66% have been proven to be catalytically active using fluorescent homogeneous assays in 384-well format compatible with the high-throughput screening criteria. The implications of these results are discussed in light of the application of this procedure to genomic-predicted peptidases.
authors.affiliation: Axxam s.r.l., Via Olgettina 58, 20132 Milano, Italy.
authors.forename: Loredana
authors.initials: L
authors.lastname: Redaelli
authors.forename: Francesca
authors.initials: F
authors.lastname: Zolezzi
authors.forename: Vanessa
authors.initials: V
authors.lastname: Nardese
authors.forename: Beatrice
authors.initials: B
authors.lastname: Bellanti
authors.forename: Valeria
authors.initials: V
authors.lastname: Wanke
authors.forename: Daniele
authors.initials: D
authors.lastname: Carettoni
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D004798:Enzymes / Q000096:biosynthesis* / Q000235:genetics; D024861:Genetic Enhancement / Q000379:methods*; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D018411:Spodoptera / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods
chemical_list: D004798:Enzymes; D011994:Recombinant Proteins