Redaelli Loredana, Zolezzi Francesca, Nardese Vanessa, Bellanti Beatrice, Wanke Valeria, Carettoni Daniele
Axxam s.r.l., Via Olgettina 58, 20132 Milano, Italy.
J Biotechnol. 2005 Oct 17;120(1):59-71. doi: 10.1016/j.jbiotec.2005.05.026. Epub 2005 Jul 25.
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.
