Abstract

First-strand cDNA encoding a serine protease inhibitor was synthesized from RNA extracted from Hevea brasiliensis leaves, RRIM600 cultivar. A full-length cDNA of RRIM600 H. Brasiliensis protease inhibitor (600Hbpi) (GenBank accession no. KJ471471) was obtained from reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The primers for 600Hbpi were created from alignments of H. Brasiliensis RRIM600 latex protease inhibitor (Hb-PI) (GenBank accession no. EU295479) and H. Brasiliensis protease inhibitor protein 1 (PI1) (GenBank accession no. AY221985). 600HbPI encodes a 70 amino acid protein and is a member of the potato inhibitor I (PI-I) family of serine protease inhibitors. Multiple sequence alignment of homologous PI-I family proteins revealed one motif WPEL of 600HbPI conserved across the PI-I family. The coding region for the active site of 600HbPI was predicted as Met⁴⁶-Glu⁴⁷. 600Hbpi was cloned into the pFLAG-ATS vector. Recombinant 600HbPI was expressed as 11 kDa proteins in Escherichia coli strain BL21. Protease inhibition analysis showed that recombinant 600HbPI is more effective at inhibiting subtilisin A than chymotrypsin but did not inhibit trypsin protease. These results indicate that the recombinant 600HbPI encoded a functional protease inhibitor that specifically targets the chymotrypsin and subtilisin classes of serine proteases.