Nucleotide sequence and expression in Escherichia coli of cDNAs encoding papaya proteinase omega from Carica papaya.
We have cloned and sequenced two similar, but distinct, cDNAs from both fruit and leaf tissues of Carica papaya. The C-terminal portion of the predicted amino acid (aa) sequence of one of the clones has complete identity with the mature enzyme sequence of the cysteine proteinase papaya proteinase omega (Pp omega). The second clone contains ten individual bp changes compared with the first and encodes a protein with three single-aa substitutions, only one of which is located in the mature sequence, but most noticeably carries an additional 19-aa C-terminal extension. The clones encode pre-pro precursor isoforms of Pp omega. The former of these clones has been expressed in Escherichia coli using a T7 polymerase expression system to produce insoluble pro-enzyme which has been solubilized and refolded to yield auto-activable pro-Pp omega.
Revell
D F
DF
Department of Protein Engineering, AFRC Institute of Food Research, Reading Laboratory, UK.
Cummings
N J
NJ
Baker
K C
KC
Collins
M E
ME
Taylor
M A
MA
Sumner
I G
IG
Pickersgill
R W
RW
Connerton
I F
IF
Goodenough
P W
PW
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular / Q000379:methods; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010940:Plant Proteins; D010944:Plants / Q000201:enzymology* / Q000235:genetics*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification
D007527:Isoenzymes; D015345:Oligonucleotide Probes; D010940:Plant Proteins; D012313:RNA; D004247:DNA; D003546:Cysteine Endopeptidases; C039974:caricain