Secretion of functional papain precursor from insect cells. Requirement for N-glycosylation of the pro-region.
The synthetic gene coding for the precursor of the cysteine protease papain (EC 3.4.22.2) has been expressed using the baculovirus/insect cell system. The prepropapain gene was cloned into the transfer vector IpDC125 behind the polyhedrin promoter. The recombinant construct was then incorporated by homologous recombination into the Autographa californiaca nuclear polyhedrosis virus genome. The host Spodoptera frugiperda Sf9 cells infected with the recombinant baculovirus secrete an enzymatically inactive N-glycosylated papain precursor. This zymogen could be activated in vitro to yield about 400 nmol of active papain per liter of culture. The recombinant active mature papain was enzymatically indistinguishable from natural papain but the precursor was not processed to the same amino acid residue. The insect cells also accumulated prepropapain and glycosylated propapain intracellularly. This accumulation was an indication that there are rate-limiting steps in the secretion of proteins from insect cells in this expression system. Characterization of mutants of the precursor has shown that entry into the secretory pathway and addition of carbohydrate are prerequisite conditions for the production and secretion of functional propapain.
Genetic Engineering Section, National Research Council of Canada, Montreal, Quebec.
T
T
Vernet
D C
DC
Tessier
C
C
Richardson
F
F
Laliberté
H E
HE
Khouri
A W
AW
Bell
A C
AC
Storer
D Y
DY
Thomas
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics*; D005813:Genes, Synthetic; D006031:Glycosylation; D007304:Insect Viruses / Q000235:genetics; D008969:Molecular Sequence Data; D009036:Moths; D009154:Mutation; D015345:Oligonucleotide Probes; D010206:Papain / Q000096:biosynthesis / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis
D004792:Enzyme Precursors; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D010206:Papain