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Planta ; 245(2): 343-353, 2017 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-27778107

RESUMEN

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.


Asunto(s)
Maclura/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Semillas/química , Inhibidores de Tripsina/metabolismo , Clonación Molecular , Modelos Moleculares , Mapeo Peptídico , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Conformación Proteica , Homología de Secuencia de Aminoácido , Tripsina/metabolismo , Inhibidor de la Tripsina de Soja de Bowman-Birk , Inhibidores de Tripsina/química , Inhibidores de Tripsina/aislamiento & purificación
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