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

• Published in: Plant science (Limerick) 1999-07, Vol.145 (1), p.41-47
• Main Authors: Taylor, Mark A.J., Al-sheikh, Maha, Revell, Dean F., Sumner, Ian G., Connerton, Ian F.
• Format: Article
• Language: English
• Subjects: amino acid sequences; Biological and medical sciences; Carica papaya; Chymopapain; complementary DNA; Cysteine proteinases; enzyme activity; Enzymes; Escherichia coli; Fundamental and applied biological sciences. Psychology; genbank/aj131988; genbank/aj131995; genbank/aj131996; genbank/aj131997; genbank/x97789; isozymes; leaves; Metabolism; molecular conformation; molecular sequence data; nucleotide sequences; Plant physiology and development; plant proteins; Prochymopapain isoforms; protein synthesis; recombinant proteins; zymogens
• ISSN: 0168-9452; 1873-2259
• DOI: 10.1016/S0168-9452(99)00068-0

Chymopapain is one of the four known cysteine proteinases found in the latex of Carica papaya. DNA sequencing of clones derived from a leaf cDNA library identified five cDNA types coding for precursor chymopapains. All of these 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 apparatus. Another amino acid substitution found in two of the isoforms at position 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 chymopapain that had comparable kinetic constants to authentic native enzyme.