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

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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.

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Acknowledgments

We are grateful to Dr. C. S. Nautiyal, Director, CSIR-NBRI, Lucknow, for providing infrastructural support and Dr. Vinod Bhakuni, CSIR-CDRI, Lucknow, for providing facility for optical spectroscopy. We thankfully acknowledge the Council of Scientific and Industrial Research (C.S.I.R.), New Delhi, India, for providing fellowship to SJ and SA. This work was carried out under the CSIR-NBRI In-house Project OLP 0031. SJ also thankfully acknowledges DST-SERB for Young Scientist grant (SB/YS/LS-39/2014), UGC for Start-up research grant (F.30-50/2014-BSR) and UGC-CAS program in department of Botany, J.N.V. University, Jodhpur.

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Authors and Affiliations

  1. 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

    Shweta Jha, Saurabh Agarwal, Indraneel Sanyal & D. V. Amla

  2. Department of Botany (Centre of Advanced Study), Jai Narain Vyas University, Jodhpur, 342001, India

    Shweta Jha

  3. Baylor College of Medicine, Houston, TX, USA

    Saurabh Agarwal

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  1. Shweta Jha
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  2. Saurabh Agarwal
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  3. Indraneel Sanyal
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  4. D. V. Amla
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Correspondence to Shweta Jha.

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Pretreatment of leaf extracts of transgenic tomato plants by (NH4)2SO4 precipitation (salting-out). (PDF 61 kb)

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Jha, S., Agarwal, S., Sanyal, I. et al. Single-Step Purification and Characterization of A Recombinant Serine Proteinase Inhibitor from Transgenic Plants. Appl Biochem Biotechnol 179, 220–236 (2016). https://doi.org/10.1007/s12010-016-1989-8

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  • DOI: https://doi.org/10.1007/s12010-016-1989-8

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