The use of mammalian cassette vector, pDR-OriP-Fc1 allowed the rapid conversion as described above.
A mouse scFv library was constructed using phagemid vector, pDR-D1, and after cell panning against CD9 transfectant, the enriched scFv repertoire was directly transferred to mammalian cassette vector, pDR-OriP-Fc1.
After cell panning on the CD transfectant, the enriched scFv repertoire in phagemid vector, pDR-D1 was transferred into mammalian cassette vector, pDR-OriP-Fc1 simply by cut and paste restriction fragment cloning.
A phagemid vector, pDR-D1 was used for cloning and display of antibody repertoire as described previously [ 16].
A mouse scFv library constructed by using a phagemid vector, pDR-D1 was subjected to cell panning against stable CD9 transfectant, and the scFv repertoire from the enriched phage pool was directly transferred to a mammalian cassette vector, pDR-OriP-Fc1.
For the coexpression assays with interactors in yeast, putative interactors were inserted, by LR reaction, in the yeast expression vector pDR-XN-GW vector, which replaced URA3 with LEU2 in pDRf1 containing the f1 replication origin, GATEWAY cassette (-attR1- CmR- ccdB-attR2), PMA1 promoter fragment, and ADH terminator in yeast (Loqué et al., 2007).
Here we used two vectors, pDR-D1 for phage display of scFv and pDR-OriP-Fc1 for mammalian expression of scFv-Fc.
The yeast strain 22Δ8AA [7] was transformed with pDR vector containing the cDNA of CAT8 or empty vector pDR196 by heat shock.
Positive clones for each element along with the pDR vector were transfected in P. falciparum by electroporation.
For rapid conversion of phage displayed scFv into Fc-fusion form, a mammalian expression vector, pDR-OriP-Fc1 was used as described previously [ 16].
