Cloning of Corn Cystatin cDNA and Evaluation of Its Protein Inhibitory Effect in vitro

Isolating and cloning of plant protease inhibitor (PIs) genes and transforming them to the genome of other plants have paved the way for producing resistant transgenic plants against pests. Knowing that cystatins act as inhibitor factor against cysteine protease, short and long cystatin genes were isolated from maize mRNA. By using specific primers, cDNA of these genes were constructed and cloned in pUC19 and pGEX 2T plasmid vectors. The recombinant plasmid vectors were then transformed to E. coli (strain DH5 α) competent cells using electroporation. The competent cells harboring the clones were grown in suitable medium and cystatin proteins fused to Glutation-S-transferase (GST) were purified by glutation agarose bead filter. In each step of the procedure the presence of cystatin genes were confirmed through electrophoresis. Further evaluation proved the inhibitory effect and a mild stability of at least one of the corn cystatin (CC II) when incubated with cysteine protease.