The baculovirus expression vector pBSV-8His directs secretion of histidine-tagged proteins.
A novel baculovirus expression vector (pBSV-8His) directs secretion of recombinant proteins into the culture medium of infected insect cells. By providing a vector-encoded signal peptide upstream from a multiple cloning site, the product of the inserted cDNA is directed to the secretory pathway. In addition, a C-terminal His-tag allows convenient purification of the native protein directly from the culture medium in less than 5 h. The His-tag can be cleaved off the purified protein by utilizing an enterokinase cleavage site located directly N-terminal to the His sequence. By insertion of a coding sequence representing the human complement regulatory factor H-like (FHL-1) plasma protein into pBSV-8His, a high level of protein synthesis was demonstrated (9 micrograms/ml). The high level of production and the ease with which native protein can be purified almost to homogeneity, makes pBSV-8His particularly suitable for protein synthesis and purification. The combination of a vector-encoded signal peptide and a C-terminal His-tag allows production and direct purification of individual domains of secretory proteins in eukaryotic cells. This feature makes this novel vector extremely useful for structure function analyses of secretory proteins.
Kühn
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Department of Molecular Biology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
Zipfel
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PF
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors; D006639:Histidine; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009532:Nickel; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D018411:Spodoptera
D011994:Recombinant Proteins; D006639:Histidine; D009532:Nickel