EXTRACTION, PURIFICATION, AND CHARACTERIZATION OF A TRYPSIN INHIBITOR FROM COWPEA SEEDS (Vigna unguiculata)

Abstract

Protease inhibitors against trypsin were extracted from cowpea seeds, purified, and characterized. After the seed powder was defatted with hexane, the cowpea trypsin inhibitor (CpTI) was extracted with 0.15 M NaCl for 30 min. The crude extracts were then heated at 90°C for 10 min, followed by precipitation with 40-65% saturation ammonium sulfate, by which the protein purity increased approximately 15-fold. The CpTI had approximate 88-fold and 186-fold purification after anion-exchange chromatography (Super-Q) and gel filtration (Sephadex G-200), respectively. A broad band of the purified CpTI on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicates a degree of heterogeneity and partial denaturation of CpTI, having a molecular mass of ∼8000 kD. Multiple peaks between 7451 and 8898 by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy also suggest heterogeneity. The purified CpTI was stable at 90°C for 60 min, pH 5-10, and 0-3.0% of NaCl. The purification method described here can be used to obtain highly purified CpTI for its studies such as risk assessment of CpTI genetically modified foods.

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

“…Interestingly, the nucleotide sequence has some extra six amino acids at the C terminus (VMPHQQ) versus the purified inhibitor sequence. The presence of multiple genes and the possibility of hydrolysis support the idea that a large number of isoinhibitors can be present and coexist within M. pomifera seeds (Kalume et al 1995;Wang et al 2014).…”

Biological Activity of Serine Protease Inhibitor Isolated from the Seeds of Phaseolus vulgaris

“…However, trypsin inhibitor was purified to 13.51-fold with 30.53% recovery from seed flour of Vigna radiata L. Wilczek by ammonium sulphate precipitation and gel filtration chromatography on Sephadex G-75. Higher fold purification was achieved from seeds flour of P. vulgaris L., Albizia lebbeck and cowpea (Vigna unguiculata) because ion-exchange chromatography in addition to above mentioned steps for purification was used [4,18,22]. Native-PAGE and SDS-PAGE of partially purified inhibitor protein from Baspa cultivar revealed two protein bands.…”

Neutrophil elastase inhibitor purification strategy from cowpea seeds

“…Additional inhibitors were purified in Vigna unguiculata , using different processes such as CpTI trypsin inhibitor, purified by ammonium sulfate precipitation, followed by anion-exchange chromatography (Super-Q) and gel filtration (Sephadex G-200). CpTI has approximately 8 kDa and thermal stability (up to 90°C) [35]. Another inhibitor identified in the Vigna unguiculata is the CpPI (in two cowpea cultivars: Cream7 and Buff), purified by ammonium sulfate fractionation and DEAE-Sephadex A-25 column.…”