Secretion and purification of recombinant beta1-4 galactosyltransferase from insect cells using pFmel-protA, a novel transposition-based baculovirus transfer vector.
The palette of transfer vectors available for generation of recombinant baculoviruses based on transposition-mediated recombination has been enlarged by constructing the pFmel-protA vector. The pFmel-protA plasmid includes the honeybee melittin secretion signal and a Staphylococcus aureus protein A fusion protein tag, which allows the secretion and purification of recombinant proteins. Using this system, the human beta1-4 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 recombinant protein expression by enzyme-linked immunosorbent assay and Western blotting. Single step purification was achieved by immunoglobulin G affinity chromatography achieving a recovery yield of 28% and a specific activity of 1.9 U per mg of recombinant protein.
Zhou
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Institute of Physiology, University of Zürich, Winterthurerstrasse 190, Zürich, CH-8057, Switzerland.
Malissard
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Berger
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EG
Hennet
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D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D001516:Bees; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017077:Culture Media, Conditioned / Q000737:chemistry; D004251:DNA Transposable Elements / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors / Q000235:genetics; D007313:Insecta / Q000166:cytology / Q000201:enzymology*; D008555:Melitten / Q000235:genetics*; D008969:Molecular Sequence Data; D009237:N-Acetyllactosamine Synthase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000235:genetics
D017077:Culture Media, Conditioned; D004251:DNA Transposable Elements; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D008555:Melitten; D009237:N-Acetyllactosamine Synthase