Single-Step Purification and Characterization of A Recombinant Serine Proteinase Inhibitor from Transgenic Plants

Expression of recombinant therapeutic proteins in transgenic plants has a tremendous impact on safe and economical production of biomolecules for biopharmaceutical industry. The major limitation in their production is downstream processing of recombinant protein to obtain higher yield and purity of the final product. In this study, a simple and rapid process has been developed for purification of therapeutic recombinant alpha(1)-proteinase inhibitor (r alpha(1)-PI) from transgenic tomato plants, which is an abundant serine protease inhibitor in human serum and chiefly inhibits the activity of neutrophil elastase in lungs. We have expressed r alpha(1)-PI with modified synthetic gene in transgenic tomato plants at a very high level (a parts per thousand integral 3.2 % of total soluble protein). The heterologous protein was extracted with (NH4)(2)SO4 precipitation, followed by chromatographic separation on different matrices. However, only immunoaffinity chromatography resulted into homogenous preparation of r alpha(1)-PI with 54 % recovery. The plant-purified r alpha(1)-PI showed molecular mass and structural conformation comparable to native serum alpha(1)-PI, as shown by mass spectrometry and optical spectroscopy. The results of elastase inhibition assay revealed biological activity of the purified r alpha(1)-PI protein. This work demonstrates a simple and efficient one-step purification of r alpha(1)-PI from transgenic plants, which is an essential prerequisite for further therapeutic development.