Heterologous expression and purification of human placental ribonuclease inhibitor

Human placental ribonuclease inhibitor (RI) is a wide spread cytoplasmic protein regulating RNase activity in vivo and extensively used in a variety of molecular biology applications in vitro where RNase contamination is a potential problem. The RI is distinguished by its unique features, such as being hydrophobic leucine-rich repeat motifs and containing a high amount of reduced cysteine residues. The expression of recombinant RI is extremely difficult to reach high levels in soluble form in the cytoplasm of Escherichia coli. Here, we utilized N-terminal fusion partner, the set of molecular chaperones, reducing agents and refolding procedures to improve the soluble expression of RI. By constructing a recombinant expression vector containing 6x-His tag, sfGFP fusion tag and TEV protease cleavege side, E. coli BL21 (DE3) was preferred as a host strain for expression. The recombinant plasmid was efficiently transformed into competent BL21 host cells. The SDS-PAGE analysis was carried out to detect the expression of protein which recovered from the inclusion bodies. A single band with a molecular mass of 78 kDa was observed. N terminal tags were removed by TEV protease digestion to generate native RI and its purification was performed with Ni-NTA agarose. The activity of RI was determined by using RNA electrophoresis.