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A Bowman–Birk protease inhibitor purified, cloned, sequenced and characterized from the seeds of Maclura pomifera (Raf.) Schneid

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Abstract

Main conclusion

A new BBI-type protease inhibitor with remarkable structural characteristics was purified, cloned, and sequenced from seeds of Maclura pomifera , a dicotyledonous plant belonging to the Moraceae family.

In this work, we report a Bowman–Birk inhibitor (BBI) isolated, purified, cloned, and characterized from Maclura pomifera seeds (MpBBI), the first of this type from a species belonging to Moraceae family. MpBBI was purified to homogeneity by RP-HPLC, total RNA was extracted from seeds of M. pomifera, and the 3′RACE-PCR method was applied to obtain the cDNA, which was cloned and sequenced. Peptide mass fingerprinting (PMF) analysis showed correspondence between the in silico-translated protein and MpBBI, confirming that it corresponds to a new plant protease inhibitor. The obtained cDNA encoded a polypeptide of 65 residues and possesses 10 cysteine residues, with molecular mass of 7379.27, pI 6.10, and extinction molar coefficient of 9105 M−1 cm−1. MpBBI inhibits strongly trypsin with K i in the 10−10 M range and was stable in a wide array of pH and extreme temperatures. MpBBI comparative modeling was applied to gain insight into its 3D structure and highlighted some distinguishing features: (1) two non-identical loops, (2) loop 1 (CEEESRC) is completely different from any known BBI, and (3) the amount of disulphide bonds is also different from any reported BBI from dicot plants.

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Abbreviations

BBI:

Bowman–Birk inhibitor

MpBBI:

Maclura pomifera Bowman–Birk inhibitor

PMF:

Peptide mass fingerprinting

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Acknowledgements

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP-Project-12/50191-4R, D.A. and M.A.J.), CYTED (Red temática PROMAL 210RT0398, C.M.L., N.O.C., F.X.A., L.M.I.L. and S.A.T.) and CONICET (PIP 0297, L.M.I.L. and S.A.T.). The MALDI-TOF MS analyses were carried out in the Proteomics and Bioinformatics Facility of the Universitat Autònoma de Barcelona (SePBioEs-UAB) by S.A.T.

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Correspondence to Laura M. I. López or Sebastián A. Trejo.

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The authors declare that they have no competing interests.

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L. M. I. López and S. A. Trejo contributed equally.

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Indarte, M., Lazza, C.M., Assis, D. et al. A Bowman–Birk protease inhibitor purified, cloned, sequenced and characterized from the seeds of Maclura pomifera (Raf.) Schneid. Planta 245, 343–353 (2017). https://doi.org/10.1007/s00425-016-2611-6

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