Construction of recombinant Pichia pastoris expressing single-chain antibody fragment against extracellular domain of EpCAM

Epithelial cell adhesion molecule (EpCAM) is highly expressed on epithelial tumors. So, EpCAM is a valuable antigen for targeted therapy. Using monoclonal antibodies (mabs) is an attractive approach for targeted cancer therapy. Importantly, limitations of intact mabs including large size led to the development of antibody fragments such as single chain fragment variable (scfv). Pichia pastoris is considered as a suitable host for large-scale production of recombinant proteins because of post-translation modification system and low cost. Here, the Pichia expressing a scfv against EpCAM extracellular domain (EpEX) was constructed.

The codon optimized gene encoding anti-EpEX-scfv protein was cloned in the XhoI and XbaI sites of the pPICZαB vector. The recombinant plasmid was confirmed by restriction enzyme analysis and sequencing. The constructed plasmid was integrated into GS115 strain by electroporation. Positive clones were evaluated by PCR using three sets of primers.

Restriction enzyme analysis utilizing HindIII and BamHI (2788, 1537 bp bands), as well as XhoI, XbaI (3506, 819 bp bands) confirmed construction of recombinant pPICZαB-anti EpEX- scfv. In this way, PCR based screening results of integrants showed two bands (2200 and 1321 bp), when AOX1 primer set was used as well as one band (968 bp) when the 3´ insert-specific primer paired with 5´ AOX1 primer. There was one band (751 bp) in PCR result of confirmed integrants when insert-specific primer set was utilized.

These findings imply that the engineered strain was constructed. The anti-EpEX-scfv protein can be used as a potential candidate in cancer immunotherapy.