A Bowman–Birk protease inhibitor purified, cloned, sequenced and characterized from the seeds of Maclura pomifera (Raf.) Schneid
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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.
KeywordsBBI-type protease inhibitor Cloning Homology modeling Loop Three-dimensional structure Trypsin inhibition
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.
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Conflict of interest
The authors declare that they have no competing interests.