Secretion of thermostable DNA polymerase using a novel baculovirus vector.
Baculovirus-mediated expression has become a powerful tool for the high yield production of functionally active recombinant proteins. In order to further enhance the utility of this expression system, we constructed versatile transfer vectors that facilitate the secretion of recombinant proteins from host insect cells by inserting functional secretory leader sequences down-stream of the polyhedrin promoter. In-frame insertion of cDNA sequences results in the synthesis of fusion proteins containing a heterologous signal sequence which directs the recombinant protein to the secretory pathway. Human and insect leader sequences were successfully tested with a number of proteins including the thermostable Pyrococcus furiousus (Pfu) DNA polymerase in an effort to maximize secretion of heterologous proteins from insect cells. The human placental alkaline phosphatase signal sequence (MLGPCMLLLLLLLGLRLQLSLG) proved to be optimal for the secretion of not only this thermostable bacterial enzyme but also for the secretion of other biologically active polypeptides.
Mroczkowski
B S
BS
Agouron Institute, La Jolla, California 92037.
Huvar
A
A
Lernhardt
W
W
Misono
K
K
Nielson
K
K
Scott
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001105:Archaea / Q000201:enzymology; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D016895:Culture Media, Serum-Free; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D005822:Genetic Vectors; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D016895:Culture Media, Serum-Free; D017931:DNA Primers; D011994:Recombinant Proteins; D004259:DNA-Directed DNA Polymerase