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Purification, Characterisation and Cloning of a 2S Albumin with DNase, RNase and Antifungal Activities from Putranjiva Roxburghii

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An Erratum to this article was published on 23 August 2014

Abstract

The present study reports the characterisation of a novel ~12-kDa heterodimeric protein, designated as putrin, from the seeds of Putranjiva roxburghii. The purification of putrin to homogeneity was accomplished using DEAE-sepharose where protein was unbound, CM-sepharose and Cibacron blue 3GA where it was bound and appeared as single peak on a size-exclusion chromatography column. A 15 % sodium dodecyl sulphate polyacrylamide electrophoresis gel, under reducing condition, demonstrated that putrin is made of two polypeptide chains of approximately 4.5 and 7.5 kDa. Circular dichroism studies demonstrated the helical nature and conformational stability of protein at increasing temperatures. Putrin exhibited both RNase and DNase activities and exerted antifungal activity but possessed relatively weak translation–inhibitory activity in cell-free system. The cloning and sequence analysis revealed a 414 bp open reading frame encoding a preproprotein of 137 amino acid residues. The amino acid sequence comparisons and phylogenetic analysis of putrin showed significant homology to 2S seed storage family proteins. The results demonstrated that putrin belongs to 2S albumin family and exhibits a spectrum of biotechnologically exploitable functions.

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Acknowledgements

This work was supported by a grant no. IRIS ID No. 2011-15280 from Indian Council of Medical Research, Government of India. CD studies and gel-exclusion chromatography were performed at NMR facility and Macromolecular Crystallographic Unit, respectively, at Institute Instrumentation Centre (IIC), IIT Roorkee. The N-terminal sequencing was performed at the protein sequencing facility of IMTECH, Chandigarh. Authors thank Dr. Partha Roy for allowing and Ms. Rajani Salunke for assisting to use luminometer in their lab. NSC and DG thanks UGC, PPST thanks CSIR, GKP thanks MHRD and PS thanks DBT, Government of India, for financial support.

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Author notes

  1. Navneet S. Chaudhary

    Present address: Department of Botany, University of Rajasthan, Jaipur, 302004, India

Authors and Affiliations

  1. Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247667, India

    Prabhat Pratap Singh Tomar, Navneet S. Chaudhary, Pushkar Mishra, Deepankar Gahloth, Girijesh Kumar Patel, Purushotham Selvakumar, Pranav Kumar & Ashwani Kumar Sharma

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  1. Prabhat Pratap Singh Tomar
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Correspondence to Ashwani Kumar Sharma.

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Tomar, P.P.S., Chaudhary, N.S., Mishra, P. et al. Purification, Characterisation and Cloning of a 2S Albumin with DNase, RNase and Antifungal Activities from Putranjiva Roxburghii . Appl Biochem Biotechnol 174, 471–482 (2014). https://doi.org/10.1007/s12010-014-1078-9

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