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

• Published in: Planta 2017-02, Vol.245 (2), p.343-353
• Main Authors: Indarte, Martín, Lazza, Cristian M., Assis, Diego, Caffini, Néstor O., Juliano, María A., Avilés, Francesc X., Daura, Xavier, López, Laura M. I., Trejo, Sebastián A.
• Format: Article
• Language: English
• Subjects: Agriculture; Biomedical and Life Sciences; Cloning, Molecular; Ecology; Forestry; Life Sciences; Liquid chromatography; Maclura - chemistry; Maclura pomifera; Models, Molecular; Moraceae; ORIGINAL ARTICLE; Peptide Mapping; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - isolation & purification; Plant Proteins - metabolism; Plant Sciences; Protein Conformation; Proteinase inhibitors; Seeds; Seeds - chemistry; Sequence Homology, Amino Acid; Species extinction; Trypsin - metabolism; Trypsin Inhibitor, Bowman-Birk Soybean; Trypsin Inhibitors - chemistry; Trypsin Inhibitors - isolation & purification; Trypsin Inhibitors - metabolism
• ISSN: 0032-0935; 1432-2048
• DOI: 10.1007/s00425-016-2611-6

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 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.