Cloning and sequencing of papain-encoding cDNA

Abstract

Messenger RNA extracted from Carica papaya fruit was converted to cDNA and cloned into the PstI restriction site of plasmid pBR322. Subclones of the approximately 1.4-kb fragment were sequenced. The nucleotide sequence matched that expected, based on the amino acid (aa) sequence for papain, with the following exceptions: at aa positions 47, 118 and 135 the codon for glutamate was found instead of glutamine; at aa position 169 the codon for asparagine was found instead of glycine; at aa positions 86-88, a difference in the order of the aa codons was observed, namely tyr-pro-tyr instead of the published pro-tyr-tyr. The upstream sequence revealed that papain is probably synthesized with a 133-aa prosegment, suggesting that the enzyme is synthesized as an inactive zymogen. The downstream segment revealed an unusual (AT)9AGAA sequence beginning 26 bp from the double TGA stop codon.