Enhanced secretion from insect cells of a foreign protein fused to the honeybee melittin signal peptide.
The baculovirus/insect cell system has been remarkably successful in yielding high levels of synthesis of many proteins which have been difficult to synthesize in other host/vector systems. The system is also capable of secreting heterologous proteins, but with generally low efficiency. We have increased the efficiency of secretion of the system by using signal peptides of insect origin to direct the secretion of a foreign protein. The precursor of the plant cysteine protease papain (propapain) has been used as a report enzyme to compare secretion efficiency. Insect cells infected with a baculovirus recombined with the gene encoding propapain fused to the sequence encoding the honeybee melittin signal peptide secreted over five times more papain precursor than the wild-type prepropapain which used the plant signal peptide. Based on these results, we have assembled pVT-Bac, an Autographa californica nuclear polyhedrosis virus transfer vector that may enhance secretion of other foreign proteins from insect cells. The vector incorporates a number of features: phage f1 ori to facilitate site-directed mutagenesis, the strong polyhedrin promoter upstream from the melittin signal peptide-encoding sequence, and eight unique restriction sites to facilitate fusion of heterologous genes.
Tessier
D C
DC
Genetic Engineering Section, National Research Council of Canada, Montréal Québec.
Thomas
D Y
DY
Khouri
H E
HE
Laliberté
F
F
Vernet
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D007313:Insecta; D007700:Kinetics; D008555:Melitten / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010206:Papain / Q000096:biosynthesis / Q000235:genetics*; D010944:Plants / Q000201:enzymology / Q000235:genetics; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D014162:Transfection
D015345:Oligonucleotide Probes; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D008555:Melitten; D010206:Papain