Cloning, expression, and mutagenesis of trypsin inhibitor ETIb from Erythrina variegata seeds

Erythrina variegata trypsin inhibitors designated ETIa and ETIb belong to the Kunitz family trypsin inhibitor, but ETIa is unique in its ability to inhibit tissue-type plasminogen activator, while ETIb is not. The cDNA clone encoding ETIb was isolated from the seed cDNA library constructed in the lambda phage lambda gt11. The ETIb cDNA insert consists of 765 bp, including an open reading frame of 606 pb from ATG to TGA codons. The deduced amino acid sequence consists of 202 amino acids, having the signal peptides of 22 amino acids in the N-terminus and 2 amino acids in C-terminus. The cDNA fragment encoding the mature form of ETIb was introduced into an expression vector, pET-22b, and expressed in Escherichia coli BL21 (ED3) in a functional form. Furthermore, the ETIb mutant bP61R/F62L, in which Pro61 and Phe62 in ETIb were changed to the corresponding amino acid residues Arg and Leu, respectively, as in ETIa, was constructed, and its inhibitory potency toward tPA was assayed. This mutant showed significant tPA inhibitory activity, albeit less than ETIa. The result demonstrates that the Arg61 and Leu62 residues in ETIa are important in inhibiting tPA, and also suggest that beside these two residues, the other amino acid(s) or other structural element may be involved in interaction of ETIa with tPA.