EXPRESSION, PURIFICATION OF RECOMBINANT APOLIPOPROTEIN E4 UNDER NON-DENATURING CONDITIONS, AND MITOCHONDRIAL MORPHOLOGY ANALYSIS IN A NEURONAL CLONAL CELL LINE

EZEQUIEL A. SERRANO; ANGEL G. VALDIVIESO; FRANCISCO J. BARRANTES

Congreso; REUNIÓN DE SOCIEDADES DE BIOCIENCIAS 2021; 2021

LXVI REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA (SAIC)

Apolipoprotein E (ApoE) is the key risk factor for Alzheimer disease.The APOE4 allele increases the probability of developing the disease up to 15 times in homozygote carriers. Among other effects,ApoE4 has been associated with alterations in mitochondrial dynamics. In order to learn about the effects of ApoE4 on neuronal cells, weaimed at purifying the recombinant protein expressed in E. coli andassaying it on mitochondria of a neuronal clonal cell line, CNh. E. coliBL21 strain expressing the ApoE4 (pET32-E43C, containing His andTrx-tags) and 3C-protease were grown in LB medium and inducedwith 1 mM of IPTG for 1.5 and 4 h at 37 and 30Cº, respectively.Crude soluble ApoE was purified by affinity chromatography using aNi-NTA resin. The 3C-protease was purified by FPLC (Superose-12size exclusion). ApoE4 and 3C-protease were incubated at 4ºC in a25:1 ratio to release the His-Trx tag. ApoE4 was next purified by sizeexclusion chromatography, with a yield of ~5 mg/L without denaturing/renaturing steps and no apparent contaminant bands in SDSpolyacrylamide gels. We next studied mitochondrial morphology inneuronal CNh cells using confocal fluorescence microscopy. Cellswere incubated with17mg/mL ApoE4 and mitochondria labelled withMitoTracker Orange and imaged in vivo. Mitochondrial morphologywas analyzed with the Mitochondrial Network Analysis plugin for Fiji.Individual counts (1 branch), networklength and mitochondrial footprint (area covered) were quantified.ApoE4 induced an increase in mitochondrial footprint (p