Av. Teixeira et al., SYNTHESIS AND EXPRESSION OF A GENE CODING FOR ERYTHRINA TRYPSIN-INHIBITOR (ETI), Biochimica et biophysica acta, N. Gene structure and expression, 1217(1), 1994, pp. 16-22
A gene coding for Erythrina trypsin inhibitor (ETI) was designed, base
d on the published N-terminal sequence of the protein, and synthesized
by an oligonucleotide-directed single strand break-repair mechanism.
Direct expression from the expression vector pBtac1 was unsuccessful.
A construct, encoding an extended methionyl N-terminal amino acid was
expressed from the vector pET12a which supplies a signal sequence for
export to the periplasm. Most of the expressed protein was located in
the cytoplasm but because the periplasm is an environment conducive to
the formation of disulphide bridges, only periplasmic protein was ext
racted. Cyanogen bromide cleavage at the sole methionyl residue remove
d the undesired amino acid residues that remained after signal sequenc
e peptidase processing. The resultant ETI was assayed against trypsin
and tissue plasminogen activator and found to have activity similar to
that of natural ETI.