A platform for high-throughput expression of recombinant human enzymes secreted by insect cells.
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.
Redaelli
Loredana
L
Axxam s.r.l., Via Olgettina 58, 20132 Milano, Italy.
Zolezzi
Francesca
F
Nardese
Vanessa
V
Bellanti
Beatrice
B
Wanke
Valeria
V
Carettoni
Daniele
D
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
D004798:Enzymes; D011994:Recombinant Proteins