Secretion of thermostable DNA polymerase using a novel baculovirus vector

• Published in: The Journal of biological chemistry 1994-05, Vol.269 (18), p.13522-13528
• Main Authors: MROCZKOWSKI, B. S, HUVAR, A, LERNHARDT, W, MISONO, K, NIELSON, K, SCOTT, B
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Archaea - enzymology; Baculoviridae - genetics; baculovirus; Base Sequence; Biological and medical sciences; Biotechnology; Cell Line; Cell physiology; Cloning, Molecular; Culture Media, Serum-Free; DNA Primers; DNA-Directed DNA Polymerase - genetics; DNA-Directed DNA Polymerase - metabolism; Enzyme Stability; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; Genetic Vectors; Hot Temperature; Humans; Insecta; Methods. Procedures. Technologies; Molecular and cellular biology; Molecular Sequence Data; Moths; Protein Processing, Post-Translational; Pyrococcus furiosus; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Secretion. Exocytosis; Vectors (cloning, transfer, expression). Insertion sequences and transposons
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(17)36862-x

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.