title: Expression and purification of recombinant apolipoprotein A-I Zaragoza (L144R) and formation of reconstituted HDL particles.
abstract: Apolipoprotein A-I Zaragoza (L144R) (apo A-I Z), has been associated with severe hypoalphalipoproteinemia and an enhanced effect of high density lipoprotein (HDL) reverse cholesterol transport. In order to perform further studies with this protein we have optimized an expression and purification method of recombinant wild-type apo A-I and apo A-I Z and produced mimetic HDL particles with each protein. An pET-45 expression system was used to produce N-terminal His-tagged apo A-I, wild-type or mutant, in Escherichia coli BL21 (DE3) which was subsequently purified by affinity chromatography in non-denaturing conditions. HDL particles were generated via a modified sodium cholate method. Expression and purification of both proteins was verified by SDS-PAGE, MALDI-TOF MS and immunochemical procedures. Yield was 30mg of purified protein (94% purity) per liter of culture. The reconstituted HDL particles checked via non-denaturing PAGE showed high homogeneity in their size when reconstituted both with wild-type apo A-I and apo A-I Z. An optimized system for the expression and purification of wild-type apo A-I and apo A-I Z with high yield and purity grade has been achieved, in addition to their use in reconstituted HDL particles, as a basis for further studies.
authors.affiliation: Laboratorio de Investigación Molecular, Hospital Universitario Miguel Servet IIS Aragón, Edificio de Consultas Externas, 50009 Zaragoza, Spain. sfiddyment.iacs@aragon.es
authors.forename: Sarah
authors.initials: S
authors.lastname: Fiddyment
authors.forename: Sílvia
authors.initials: S
authors.lastname: Barceló-Batllori
authors.forename: Miguel
authors.initials: M
authors.lastname: Pocoví
authors.forename: Angel-Luis
authors.initials: AL
authors.lastname: García-Otín
mesh_terms: D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D008075:Lipoproteins, HDL / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D011994:Recombinant Proteins; C426725:apolipoprotein A-I Zaragoza