OPTIMIZING PROTEIN PURIFICATION FOR RNA-BINDING RECOMBINANT FUSION PROTEINS

Abstract

There has been great interest in delivering short interfering RNA (siRNA) and microRNA (miRNA) for therapeutic applications. However, the delivery of small RNAs remains challenging due to its inefficient cellular uptake and instability under physiological conditions. Here, we engineered a CXCR4-targeting RNA-protein nanoplex that consists of a CXCR4-targeting single-chain variable fragment (scFv) antibody, which is fused to an RNA-binding protamine peptide (RSQSRSRYYRQRQRSRRRRRRS). To obtain a functional RNA-binding protein, the removal of external nucleic acids is essential. This study aims to optimize the purification process of RNA-binding fusion proteins to free up RNA-binding domains and study if protein/siRNA complexes could successfully protect and deliver siRNA to silence cellular genes. After testing out different nucleic-acid removal methods, the high-salt-wash assisted immobilized metal affinity column purification method showed a great reduction of bound nucleic-acid contaminants. The purified fusion proteins showed a successful complexation with siRNAs and could deliver siRNA to the targeted cells.Master of Scienc