title: Challenges in the expression of disulfide bonded, threonine-rich antifreeze proteins in bacteria and yeast.
abstract: Certain freeze-intolerant insects produce antifreeze proteins (AFPs) during overwintering including the spruce budworm (Choristoneura fumiferana) and yellow mealworm (Tenebrio molitor) AFP gene families. However, only a few of the isoforms, encoded by their multiple-copy gene families, have been characterized. When expressed in bacterial systems the insect AFPs have to be denatured and refolded in vitro, a procedure that is not uniformly successful, presumably due to the beta-helix structure and the requirement for disulfide bonds. In an attempt to overcome these difficulties, bacterial vectors and hosts that have been developed to produce soluble, folded proteins, as well as a yeast expression system (Pichia pastoris) were employed. Bacterial expression resulted in low quantities of active recombinant protein for certain isoforms. In contrast, both small and large-scale fermentation of recombinant AFP in Pichia yielded substantial protein production (100 mg/L) but functional ice binding activity of protein produced in three different transformed yeast strains (KM71, X33 or GS115) was low. Inappropriate O-linked glycosylation of the Thr-rich AFPs appeared to be partially reversed by mild chemical deglycosylation, but activity remained low. Substantial quantities, as well as activity were recovered when a fish AFP, with disulfide bonds, but without potential Thr glycosylation sites was expressed in the yeast system.
authors.affiliation: Department of Biology, Queen's University, Kingston, Ont., Canada K7L 3N6. tyshenko@biology.queensu.ca
authors.forename: Michael G
authors.initials: MG
authors.lastname: Tyshenko
authors.forename: Marc
authors.initials: M
authors.lastname: d'Anjou
authors.forename: Peter L
authors.initials: PL
authors.lastname: Davies
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Daugulis
authors.forename: Virginia K
authors.initials: VK
authors.lastname: Walker
mesh_terms: D000818:Animals; D021301:Antifreeze Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D003001:Cloning, Molecular; D001517:Coleoptera / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal / Q000235:genetics*; D009036:Moths / Q000235:genetics; D005810:Multigene Family; D010843:Pichia / Q000235:genetics; D020033:Protein Isoforms / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013712:Tenebrio / Q000235:genetics; D013912:Threonine / Q000378:metabolism*
chemical_list: D021301:Antifreeze Proteins; D004220:Disulfides; D020033:Protein Isoforms; D013912:Threonine