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

Abstract

A new BBI-type protease inhibitor with remarkable structural characteristics was purified, cloned, and sequenced from seeds of <i>Maclura pomifera</i>, a dicotyledonous plant belonging to the Moraceae family. In this work, we report a Bowman–Birk inhibitor (BBI) isolated, purified, cloned, and characterized from <i>Maclura pomifera</i> 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 <i>M. pomifera</i>, 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 Kᵢ 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.