The use of Group 3 LEA proteins as fusion partners in facilitating recombinant expression of recalcitrant proteins in E. coli.
Late embryogenesis abundant (LEA) proteins are intrinsically disordered proteins that accumulate in organisms during the development of dehydration stress tolerance and cold acclimation. Group 3 LEA proteins have been implicated in the prevention of cellular protein denaturation and membrane damage during desiccation and anhydrobiosis. We tested the ability of LEA proteins to facilitate recombinant expression of recalcitrant and intrinsic membrane proteins. Two Brassica napus Group 3 LEA proteins, BN115m and a truncated fragment of BNECP63, were fused to two target proteins identified as recalcitrant to overexpression in soluble form or outside of inclusion bodies. Fusion of a truncated peptide of BNECP63 is sufficient to provide soluble and high levels of recombinant overexpression of BNPsbS (an intrinsic membrane chlorophyll-binding protein of photosystem II light harvesting complex) and a peptide of the Hepatitis C viral polyprotein. Furthermore, fusion of the recombinant target proteins to BNECP63 or BN115 prevented irreversible heat- and freeze-induced precipitation. These experiments not only underscore the exploitation of LEA-type peptides in facilitating protein overexpression and protection, but also provide insights into the mechanism of LEA proteins in cellular protection.
Singh
Jas
J
Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food, 960 Carling Ave., Ottawa, Ontario, Canada.
Whitwill
Steve
S
Lacroix
Geneviève
G
Douglas
Jennifer
J
Dubuc
Elyse
E
Allard
Ghislaine
G
Keller
Wilf
W
Schernthaner
Johann P
JP
D029688:Brassica napus / Q000235:genetics; D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000235:genetics*; D045342:Light-Harvesting Protein Complexes / Q000096:biosynthesis* / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012995:Solubility; D014758:Viral Core Proteins / Q000096:biosynthesis* / Q000235:genetics
D045342:Light-Harvesting Protein Complexes; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; C066246:late embryogenesis abundant protein, plant; C069130:nucleocapsid protein, Hepatitis C virus