Secretion and purification of recombinant beta 1-4 galactosyltransferase from insect cells using pFmel-protA, a novel transposition-based Baculovirustransfer vector

The palette of transfer vectors available for generation of recombinant bac uloviruses based on transposition-mediated recombination has been enlarged by constructing the pFmel-protA vector. The pFmel-protA plasmid includes th e honeybee melittin secretion signal and a Staphylococcus aureus protein A fusion protein tag, which allows the secretion and purification of recombin ant proteins, Using this system, the human beta 1-4 galactosyltransferase-I protein was expressed in Sf9 insect cells at a level ranging from 22 to 28 U (4.8 to 6.0 mg)/L. The protein A tag enabled a simple monitoring of reco mbinant protein expression by enzyme-linked immunosorbent assay and Western blotting. Single step purification was achieved by immunoglobulin G affini ty chromatography achieving a recovery yield of 28% and a specific activity of 1.9 U per mg of recombinant protein. (C) 2000 Academic Press.