| Abstract: | Synthetic oligonucleotides representing all possible sequences of the N-terminal and internal amino acid sequences of the chymotrypsin inhibitor ECI from Erythrina variegata seeds were used to generate a probe specific for ECI-related sequences by the polymerase chain reaction on the E. variegata genomic DNA. A lambda phage cDNA library constructed from poly(A+) RNA from maturing seeds was screened with the ECI gene thus obtained as a probe and characterized by DNA sequencing. The cloned ECI cDNA comprised 737 nucleotides and one open reading frame that encoded a polypeptide chain of 203 amino acids including a signal peptide composed of 24 amino acids. An expression plasmid was designed for export of the recombinant inhibitor into the periplasm. For this purpose, the cDNA fragment encoding matured ECI was ligated into the NcoI and BamHI sites following the pel B signal sequence in the expression vector pET-22b and expressed in Escherichia coli BL21 (DE3). However, this attempt failed as the recombinant inhibitor caused the formation of inclusion bodies in E. coli cells as a heterologous preprotein (SR-ECI), with the pel B upstream leader. SR-ECI was made soluble and renatured by refolding and reoxidation, and subsequently processed with pronase to give rise to recombinant ECI (R-ECI) that had an extra methionine residue attached to the N-terminal amino acid of ECI. Purified R-ECI inhibited chymotrypsin almost as strongly as authentic ECI. |
