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Kunitz‐type protease inhibitors group B from Solanum palustre
Author(s) -
Speransky Anna S.,
Cimaglia Fabio,
Krinitsina Anastasya A.,
Poltronieri Palmiro,
Fasano Pasqua,
Bogacheva Anna M.,
Valueva Tatiana A.,
Halterman Dennis,
Shevelev Alexei B.,
Santino Angelo
Publication year - 2007
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.200700022
Subject(s) - trypsin , protease , chymotrypsin , biology , biochemistry , trypsin inhibitor , chemistry , enzyme
Five Kunitz protease inhibitor group B genes were isolated from the genome of the diploid non‐tuber‐forming potato species Solanum palustre. Three of five new genes share 99% identity to the published KPI – B genes from various cultivated potato accessions, while others exhibit 96% identity. Spls‐KPI‐B2 and Spls‐KPI‐B4 proteins contain unique substitutions of the most conserved residues usually involved to trypsin and chymotrypsin‐specific binding sites of Kunitz‐type protease inhibitor (KPI)‐B, respectively. To test the inhibition of trypsin and chymotrypsin by Spls‐KPI proteins, five of them were produced in E. coli purified using a Ni‐sepharose resin and ion‐exchange chromatography. All recombinant Spls‐KPI‐B inhibited trypsin; K i values ranged from 84.8 (Spls‐KPI‐B4), 345.5 (Spls‐KPI‐B1), and 1310.6 nM (Spls‐KPI‐B2) to 3883.5 (Spls‐KPI‐B5) and 8370 nM (Spls‐KPI‐B3). In addition, Spls‐KPI‐B1 and Spls‐KPI‐B4 inhibited chymotrypsin. These data suggest that regardless of substitutions of key active‐center residues both Spls‐KPI‐B4 and Spls‐KPI‐B1 are functional trypsin‐chymotrypsin inhibitors.