title: Single-step purification of two functional human apolipoprotein E variants hyperexpressed in Escherichia coli.
abstract: We have cloned, from total human liver RNA, the cDNA encoding apolipoprotein E3 (apoE3). Site-directed mutagenesis was used to obtain the cDNA encoding the apoE4 isoform, a major variant of this apolipoprotein in man. These two cDNAs were subcloned into the procaryotic expression vector pAHRS. A polyhistidine tag was added at the NH2-termini of the recombinant proteins (apoE3 and apoE4) to enable rapid purification. The resulting plasmids (pAHRS-apoE3 and pAHRS-apoE4) were introduced into the Escherichia coli strain BL21(DE3). Recombinant strains were grown at 37 degrees C in a Luria and Bertani medium and the addition of isopropyl beta-thiogalactoside resulted in the expression of large amounts of apoE protein (40.5 kDa), representing at least 15% of cellular proteins. The recombinant apoE isoforms were purified, under denaturating conditions, in one step by affinity chromatography on a Ni-chelated agarose column, yielding to about 20 mg of 96% pure protein per liter of culture. Compared to plasma apoE3 purified from human very low density lipoproteins, the two renatured recombinant apoE isoforms have the same secondary structure content, as revealed by circular dichroism measurement. Moreover, the recombinant apoE3 isoform shares similar properties for the association with lipids, compared to the human protein, indicating that the addition of the amino-terminal polyhistidine peptide does not influence the structure and the lipid binding properties of this recombinant apoE isoform. No differences in the secondary structure of recombinant apoE4 were detected, whereas this isoform presents specific reactivity with lipids. This simple and rapid procedure for the expression and the purification of functional recombinant apoE should therefore enable structural and physiological studies requiring large amounts of these apolipoproteins.
authors.affiliation: Centre du Médicament, URA CNRS 597, Nancy, France.
authors.forename: T
authors.initials: T
authors.lastname: Pillot
authors.forename: A
authors.initials: A
authors.lastname: Barbier
authors.forename: A
authors.initials: A
authors.lastname: Visvikis
authors.forename: K
authors.initials: K
authors.lastname: Lozac'h
authors.forename: M
authors.initials: M
authors.lastname: Rosseneu
authors.forename: J
authors.initials: J
authors.lastname: Vandekerckhove
authors.forename: G
authors.initials: G
authors.lastname: Siest
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D001057:Apolipoproteins E / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004274:DNA, Recombinant / Q000235:genetics; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D015151:Immunoblotting; D008081:Liposomes / Q000378:metabolism; D008099:Liver / Q000502:physiology; D008623:Mercaptoethanol / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D012313:RNA / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D013862:Thiogalactosides / Q000378:metabolism; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D001057:Apolipoproteins E; D004274:DNA, Recombinant; D008081:Liposomes; D011994:Recombinant Proteins; D013862:Thiogalactosides; D008623:Mercaptoethanol; D012313:RNA; D004134:Dimyristoylphosphatidylcholine