Publication related to RSI or an RSI staff member
Challenges in the expression of disulfide bonded, threonine-rich antifreeze proteins in bacteria and yeast.
Authors
- Tyshenko, Michael G, Tyshenko MG, Department of Biology, Queen's University, Kingston, Ont., Canada K7L 3N6. tyshenko@biology.queensu.ca
- d'Anjou, Marc, d'Anjou M,
- Davies, Peter L, Davies PL,
- Daugulis, Andrew J, Daugulis AJ,
- Walker, Virginia K, Walker VK,
YEAR OF PUBLICATION: 2006
SOURCE: Protein Expr Purif. 2006 May;47(1):152-61. doi: 10.1016/j.pep.2005.10.009. Epub 2005 Oct 27.
JOURNAL TITLE ABBREVIATION: Protein Expr Purif
JOURNAL TITLE: Protein expression and purification
ISSN: 1046-5928 (Print) 1046-5928 (Linking)
VOLUME: 47
ISSUE: 1
PAGES: 152-61
PLACE OF PUBLICATION: United States
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.
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.
LANGUAGE: eng
DATE OF PUBLICATION: 2006 May
DATE OF ELECTRONIC PUBLICATION: 20051027
DATE COMPLETED: 20070820
DATE REVISED: 20171116
MESH DATE: 2007/08/21 09:00
EDAT: 2005/11/18 09:00
STATUS: MEDLINE
PUBLICATION STATUS: ppublish
OWNER: NLM