, Volume 245, Issue 2, pp 343–353 | Cite as

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

  • Martín Indarte
  • Cristian M. Lazza
  • Diego Assis
  • Néstor O. Caffini
  • María A. Juliano
  • Francesc X. Avilés
  • Xavier Daura
  • Laura M. I. López
  • Sebastián A. Trejo
Original Article


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.


BBI-type protease inhibitor Cloning Homology modeling Loop Three-dimensional structure Trypsin inhibition 



Bowman–Birk inhibitor


Maclura pomifera Bowman–Birk inhibitor


Peptide mass fingerprinting



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.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

425_2016_2611_MOESM1_ESM.tiff (1.2 mb)
Supplementary material 1 (TIFF 1243 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Martín Indarte
    • 1
  • Cristian M. Lazza
    • 2
  • Diego Assis
    • 3
  • Néstor O. Caffini
    • 2
  • María A. Juliano
    • 3
  • Francesc X. Avilés
    • 4
  • Xavier Daura
    • 4
  • Laura M. I. López
    • 5
    • 6
  • Sebastián A. Trejo
    • 7
  1. 1.PHusisTherapeuticsSan DiegoUSA
  2. 2.Centro de Investigación de Proteínas Vegetales, Facultad de Ciencias ExactasUniversidad Nacional de La PlataLa PlataArgentina
  3. 3.Departamento de BiofisicaUniversidade Federal de São PauloSão PauloBrazil
  4. 4.Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de BarcelonaBarcelonaSpain
  5. 5.Instituto de Ciencias de la Salud, Universidad Nacional Arturo JauretcheBuenos AiresArgentina
  6. 6.CITECBuenos AiresArgentina
  7. 7.Laboratorio de Neurofisiología del Instituto Multidisciplinar de Biología Celular (IMBICE), La PlataBuenos AiresArgentina

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