Construction of phage-displayed library based on the lipocalin scaffold and screening anticalins with specificity for carbofuran

Carbofuran, which is used as toxic carbonate pesticide of crops, is highly toxic and small molecule. To detect the remainder of carbofuran efficiently is important to our healthy. The lipocalins, a family of diverse proteins that normally serve for the storage or transport of mostly hydrophobin and/or chemically sensitive organic compounds, can be reshaped by combinatorial protein design such that it recognizes small molecules. Sixteen residues at the center of binding site of the bilin-binding protein (BBP) from Pieris brassicae were subjected to random mutagenesis by overlapping PCR with 12 primers. Phagemid expression vector of pCANTAB5E was used to construct the lipocalin library. The library repotire is 4.0 x 10(9). BSA and OVA conjugated carbofuran respectively as the targeted protein, the library was screened with column and plate as supporter. After 3 rounds of panning, 10 phage clones were random selected with competitive elution. Dot-blotting showed that phage displayed anticalin, named as K7, bound carbofuran specifically among them. K7 can be used as potential anticalin to detect carbofuran of crops.