Expression,Purification and Activity of the Wild and Mutants of Potato I Type Protease Inhibitor rBTI

Buckwheat trypsin inhibitor(BTI) is a serine protease inhibitor of the potato inhibitor I family.BTI contains a binding loop at the molecular surface,and the P1′,P2,P6′and P8′residues are highly conserved for the formation of internal hydrogen bonds.The pExSecI-Bti-P44T and pExSecI-Bti-W53R plasmids were constructed based on crystal data of recombinant buckwheat trypsin inhibitor(rBTI) and rBTI-trypsin complex.The proteins expressed by E.coli BL21(DE3) host cells were purified with Resource Q anion exchange chromatography and a Superdex G 75 HR 10/300 gel column.The proteasome inhibition of the purified rBTI and mutants were tested in HepG2 cells.The results showed that the Ki of rBTI-P44T was 2.91 × 10-9 mol /L,and that of the wide type is 3.56 × 10-8 mol / L.However,the Ki of rBTI-W53R was 2.97 × 10-7 mol / L,which was higher than that of rBTI.Our rBTI and the two mutants were all able to inhibit the proteasomes and suppressed HepG2 cell proliferation.These results suggested that the inhibitory effect of rBTI on tumor proliferation retained,even when its anti-trypsin activity was modified by change the conserved amino acid residues.