Engineering a Recombinant Protein Platform for Targeted Delivery of Nanoparticle Therapies.

摘要

The specific targeting of nanoparticles to diseased sites and tumors remains one of the most important and challenging problems in drug delivery, but the relationship between receptor/ligand binding properties and adhesion of colloidal particles hasn't been thoroughly revealed. In order to better understand the mechanism, here we exerted efforts to create a platform for producing recombinant targeting receptors in high yield. Specifically, we have created single chain antibodies (scFv) fused to fluorescent protein tags in a mammalian cell expression system. First, we successfully constructed pEE14-based plasmids for 4-4-20 scFv with eGFP and mCherry, respectively, through three rounds of digestion and ligation. The construct has been demonstrated as a convenient and efficient platform for subcloning. Second, we found that the construct of 4420-mCherry was more robust than 4420-eGFP, when transfecting and selecting for stable cell lines. Third, we obtained ∼70 microg 4420-mCherry protein from 20 ml CHO cell cultures. Compared to our previous work using a yeast expression system where we were only able to produce 50 ug 4420 scFv from 1 L cultures, we significantly improved the yield by approximately 40 fold. In conclusion, the results suggest that our plasmid construct and protein production system are very promising and robust, thus could be utilized as a platform to produce scFv and other proteins for drug delivery research.