Surface expression and ligand-based selection of cDNAs fused to filamentous phage gene VI.
We describe a novel phage display system that affords the surface expression and hence affinity selection of cDNAs. The strategy is based on a new approach to functionally display proteins on filamentous phage through the attachment to the C-terminus of the minor coat protein VI. The utility of the method was evaluated using a cDNA library derived from the parasite Ancylostoma caninum. cDNA sequences were fused in each of the three reading frames to the 3'-end of the M13 gene VI expressed by a phagemid vector. Phages rescued from this cDNA expression library were subjected to biopanning against two serine proteases, trypsin and the human coagulation factor Xa. This led to the identification of cDNAs encoding novel members of two different families of serine protease inhibitors. The authenticity of the cDNA selected with trypsin as the target was demonstrated by purifying the encoded potent Kunitz-type inhibitor from an Ancylostoma caninum extract. The rapid isolation of specific cDNAs with the protein VI monovalent display system should facilitate the search for novel biologically important ligands.
Jespers
L S
LS
Corvas International N.V., Gent, Belgium.
Messens
J H
JH
De Keyser
A
A
Eeckhout
D
D
Van den Brande
I
I
Gansemans
Y G
YG
Lauwereys
M J
MJ
Vlasuk
G P
GP
Stanssens
P E
PE
D000595:Amino Acid Sequence; D000722:Ancylostoma / Q000201:enzymology / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D019921:Biotinylation; D015153:Blotting, Western; D002213:Capsid / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D015951:Factor Xa / Q000378:metabolism; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics; D012697:Serine Endopeptidases / Q000235:genetics; D015842:Serine Proteinase Inhibitors / Q000235:genetics; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
D018076:DNA, Complementary; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; D000516:alpha-Amylases; D012697:Serine Endopeptidases; D014357:Trypsin; D015951:Factor Xa