title: Single-Step Purification and Characterization of A Recombinant Serine Proteinase Inhibitor from Transgenic Plants.
abstract: 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 α1-proteinase inhibitor (rα1-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 rα1-PI with modified synthetic gene in transgenic tomato plants at a very high level (≃3.2 % of total soluble protein). The heterologous protein was extracted with (NH4)2SO4 precipitation, followed by chromatographic separation on different matrices. However, only immunoaffinity chromatography resulted into homogenous preparation of rα1-PI with 54 % recovery. The plant-purified rα1-PI showed molecular mass and structural conformation comparable to native serum α1-PI, as shown by mass spectrometry and optical spectroscopy. The results of elastase inhibition assay revealed biological activity of the purified rα1-PI protein. This work demonstrates a simple and efficient one-step purification of rα1-PI from transgenic plants, which is an essential prerequisite for further therapeutic development.
authors: Plant Transgenic Lab, Genetics and Molecular Biology Division, CSIR-National Botanical Research Institute, Rana Pratap Marg, P.O. Box 436, Lucknow, 226001, U.P., India. jha.shweta80@gmail.com.|Department of Botany (Centre of Advanced Study), Jai Narain Vyas University, Jodhpur, 342001, India. jha.shweta80@gmail.com.
authors: Shweta
authors: S
authors: Jha
authors: Plant Transgenic Lab, Genetics and Molecular Biology Division, CSIR-National Botanical Research Institute, Rana Pratap Marg, P.O. Box 436, Lucknow, 226001, U.P., India.|Baylor College of Medicine, Houston, TX, USA.
authors: Saurabh
authors: S
authors: Agarwal
authors: Plant Transgenic Lab, Genetics and Molecular Biology Division, CSIR-National Botanical Research Institute, Rana Pratap Marg, P.O. Box 436, Lucknow, 226001, U.P., India.
authors: Indraneel
authors: I
authors: Sanyal
authors: Plant Transgenic Lab, Genetics and Molecular Biology Division, CSIR-National Botanical Research Institute, Rana Pratap Marg, P.O. Box 436, Lucknow, 226001, U.P., India.
authors: D V
authors: DV
authors: Amla
mesh_terms: D002846:Chromatography, Affinity; D006801:Humans; D018551:Solanum lycopersicum / Q000737:chemistry / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin
keywords: Downstream processing; Heterologous protein expression; Molecular pharming; Protein purification; Recombinant therapeutic proteins; Serine proteinase inhibitor; Transgenic tomato plants