CDNA cloning and expression of Carica papaya prochymopapain isoforms in Escherichia coli

Chymopapain is one of the four known cysteine proteinases found in the late x of Carica papaya. DNA sequencing of clones derived from a leaf cDNA libra ry identified five cDNA types coding for precursor chymopapains. All of the se isoforms have a free cysteine residue at position 117, characteristic of chymopapain. Two of the isoforms possess a further free cysteine residue, which is not likely to be involved in disulphide bonds or the active site a pparatus. Another amino acid substitution found in two of the isoforms at p osition 133 is predicted to lie in the S2 subsite of the substrate binding cleft. One of the prochymopapain isoforms was expressed in Escherichia coli . Protein was expressed as insoluble inclusion body material. This protein was solubilised, refolded and autocatalytically cleaved to yield mature chy mopapain that had comparable kinetic constants to authentic native enzyme. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.