Secretion of biologically active leech hirudin from baculovirus-infected insect cells.
The thrombin inhibitor, hirudin, from the leech Hirudo Medicinalis, is the most powerful natural anticoagulant known. It has been characterized as a polypeptide of 65 amino acids which exhibits its anticoagulant properties by binding tightly and specifically to alpha-thrombin. The potency and specificity of hirudin have generated interest on its possible use in the treatment or prophylaxis of various thrombotic diseases. We have used the baculovirus expression system to efficiently produce active hirudins in insect cells. The Autographa californica nuclear polyhedrosis virus has proved useful as a helper-independent viral expression vector for high-level production of recombinant proteins in cultured insect cells. Hirudin variants (HV1 and HV2) were produced in infected insect cells as secreted proteins by joining their coding sequences to the leader peptide sequence of the vescicular stomatitis virus G protein. The recombinant products were biologically active and, interestingly, N-terminal sequencing of HV1 revealed that the heterologous leader peptide is correctly removed.
Benatti
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L
Department of Biotechnology, Farmitalia Carlo Erba, Milan, Italy.
Scacheri
E
E
Bishop
D H
DH
Sarmientos
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P
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006629:Hirudins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007865:Leeches / Q000235:genetics; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D009036:Moths; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics; D014759:Viral Envelope Proteins / Q000235:genetics
C028391:G protein, vesicular stomatitis virus; D006629:Hirudins; D008562:Membrane Glycoproteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins