Expression and purification of bovine pancreatic RNase A

The procedure to isolate and purify calf pancreatic RNase A by biochemical methods is complicated, less efficient, and not suitable for gene plasmid production. This work will utilize the bacterial expression method to solve the above difficulties. Total RNA was isolated from bovine pancreas. cDNA encoding calf RNase A was amplified by RT-PCR, subcloned into pGEM-T, and verified by sequencing. The target gene was subcloned into the expression vector pET32a. After bacterial culture, the fusion protein with Trx tag was obtained. The supernatant from the lysate was denatured with 8mol/L urea, followed by purification through Ni-column. Purified target protein was obtained after the tag was digested with Enterokinase in the column. Purified target protein was then diluted and tested for enzymatic activity. A 16KD protein was purified and verified by SDS-PAGE electrophoresis. This RNase A displays an excellent enzymatic activity to degrade RNA.