title: Bacterial expression and characterization of rat apolipoprotein E.
abstract: Apolipoprotein (apo) E is a protein involved in both lipid metabolism and neuroprotection. Recently, it has been suggested that apoE may play a role in the regulation of food intake and body weight in rodents. However, rodent plasma apoE is difficult to purify in reasonable amounts due to numerous time-consuming steps. To circumvent this, we created a bacterial expression system for the efficient production of large amounts of rat apoE. We inserted rat apoE DNA into the pET30 expression vector and overexpressed the proteins in Escherichia coli strain BL21 (DE3). A histidine tag present at the N-terminus allowed for easy purification of the recombinant protein. The tag was removed with an IgA protease (Igase) from Neisseria gonorrhoeae leaving the mature form of the protein. The use of Igase was important as several more common proteases routinely cleave apolipoproteins at undesired sites. The recombinant protein was then compared both structurally and functionally to rat plasma apoE. This expression system will be highly useful for probing the ability of rat apoE to mediate food intake in rats.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45267, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Pearson
authors.forename: Min
authors.initials: M
authors.lastname: Liu
authors.forename: Ling
authors.initials: L
authors.lastname: Shen
authors.forename: Patrick
authors.initials: P
authors.lastname: Tso
authors.forename: W Sean
authors.initials: WS
authors.lastname: Davidson
mesh_terms: D000818:Animals; D001057:Apolipoproteins E / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D008099:Liver / Q000378:metabolism; D016296:Mutagenesis; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics; D012697:Serine Endopeptidases / Q000378:metabolism; D013997:Time Factors
chemical_list: D001057:Apolipoproteins E; D018076:DNA, Complementary; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C021433:IgA-specific serine endopeptidase