Quick purification of recombinant adeno-associated viruses with the receptor-binding capture

Abstract:

[Objective] To establish a receptor-binding capture method for recombinant adeno-associated virus (rAAV) purification. [Methods] We expressed polycystic kidney disease (PKD) domains 1 and 2 of AAV receptor in E. coli as an elastin-like polypeptide (ELP) fusion protein. We purified the fusion protein by inverse transition cycling (ITC). We generated two versions of rAAV-GFP and incubated them with ELP-PKD protein. We recovered the protein-bound rAAV-GFP by ITC and extracted the viral DNA for PCR analysis. We optimized the conditions for rAAV-GFP purification and identified the purified rAAV by electron microscopy and Western blotting. [Results] ELP-PKD fusion protein was correctly expressed as a soluble protein which was purified to more than 90% purity. We demonstrated the specific affinity of ELP-PKD fusion protein for rAAV-GFP binding. We purified rAAV-GFP with 58% recovery from insect cells or 56% recovery from AAV-293 cells. After elution, we obtained final rAAV-GFP recovery rates of 46% and 44% from the two cell types, respectively. We demonstrated that the purified rAAV-GFP had the typical morphology and structural proteins of AAV. [Conclusion] We established ELP-PKD-binding capture method for quick purification of rAAV from different cell types.

Key words:adeno-associated virus receptor;ELP fusion expression;receptor-binding capture;recombinant AAV purification