title: Expression and purification of sea raven type II antifreeze protein from Drosophila melanogaster S2 cells.
abstract: We present a system for the expression and purification of recombinant sea raven type II antifreeze protein, a cysteine-rich, C-type lectin-like globular protein that has proved to be a difficult target for recombinant expression and purification. The cDNAs encoding the pro- and mature forms of the sea raven protein were cloned into a modified pMT Drosophila expression vector. These constructs produced N-terminally His(6)-tagged pro- and mature forms of the type II antifreeze protein under the control of a metallothionein promoter when transfected into Drosophila melanogaster S2 cells. Upon induction of stable cell lines the two proteins were expressed at high levels and secreted into the medium. The proteins were then purified from the cell medium in a simple and rapid protocol using immobilized metal affinity chromatography and specific protease cleavage by tobacco etch virus protease. The proteins demonstrated antifreeze activity indistinguishable from that of wild-type sea raven antifreeze protein purified from serum as illustrated by ice affinity purification, ice crystal morphology, and their ability to inhibit ice crystal growth. This expression and purification system gave yields of 95 mg/L of fully active mature sea raven type II AFP and 9.6 mg/L of the proprotein. This surpasses all previous attempts to express this protein in Escherichia coli, baculovirus-infected fall armyworm cells and Pichia pastoris and will provide sufficient protein for structural analysis.
authors.affiliation: Department of Biochemistry and the Protein Engineering Network Centres of Excellence, Queen's University, Kingston, Ont., Canada. scottera@post.queensu.ca
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Scotter
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Kuntz
authors.forename: Michelle
authors.initials: M
authors.lastname: Saul
authors.forename: Laurie A
authors.initials: LA
authors.lastname: Graham
authors.forename: Peter L
authors.initials: PL
authors.lastname: Davies
authors.forename: David R
authors.initials: DR
authors.lastname: Rose
mesh_terms: D000818:Animals; D021341:Antifreeze Proteins, Type II / Q000096:biosynthesis* / Q000302:isolation & purification*; D002460:Cell Line; D002846:Chromatography, Affinity; D004331:Drosophila melanogaster; D011498:Protein Precursors / Q000096:biosynthesis* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification*
chemical_list: D021341:Antifreeze Proteins, Type II; D011498:Protein Precursors; D011994:Recombinant Proteins; C089666:antifreeze protein, sea raven