title: A Bowman-Birk protease inhibitor purified, cloned, sequenced and characterized from the seeds of Maclura pomifera (Raf.) Schneid.
abstract: 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-1cm-1. MpBBI inhibits strongly trypsin with Kiin the 10-10M 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.
authors: PHusisTherapeutics, 3210 Merryfield Row, San Diego, CA, 92121, USA.
authors: Martín
authors: M
authors: Indarte
authors: Centro de Investigación de Proteínas Vegetales, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 1900, La Plata, Argentina.
authors: Cristian M
authors: CM
authors: Lazza
authors: Departamento de Biofisica, Universidade Federal de São Paulo, Vila Clementino, São Paulo, 04044-020, Brazil.
authors: Diego
authors: D
authors: Assis
authors: Centro de Investigación de Proteínas Vegetales, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 1900, La Plata, Argentina.
authors: Néstor O
authors: NO
authors: Caffini
authors: Departamento de Biofisica, Universidade Federal de São Paulo, Vila Clementino, São Paulo, 04044-020, Brazil.
authors: María A
authors: MA
authors: Juliano
authors: Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona, Bellaterra, 08193, Barcelona, Spain.
authors: Francesc X
authors: FX
authors: Avilés
authors: Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona, Bellaterra, 08193, Barcelona, Spain.
authors: Xavier
authors: X
authors: Daura
authors: Instituto de Ciencias de la Salud, Universidad Nacional Arturo Jauretche, 1888 Florencio Varela, Buenos Aires, Argentina. lmilopez@biol.unlp.edu.ar.|CITEC, Gonnet, B1897, Buenos Aires, Argentina. lmilopez@biol.unlp.edu.ar.
authors: Laura M I
authors: LM
authors: López
authors: Laboratorio de Neurofisiología del Instituto Multidisciplinar de Biología Celular (IMBICE), La Plata, B1906APO, Buenos Aires, Argentina. strejo@imbice.gov.ar.
authors: Sebastián A
authors: SA
authors: Trejo
mesh_terms: D003001:Cloning, Molecular; D031624:Maclura / Q000737:chemistry*; D008958:Models, Molecular; D010449:Peptide Mapping; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D012639:Seeds / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000378:metabolism; D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D014361:Trypsin Inhibitors; D014357:Trypsin
keywords: BBI-type protease inhibitor; Cloning; Homology modeling; Loop; Three-dimensional structure; Trypsin inhibition