Expression, purification, and preliminary characterization of human presenilin-2

Abstract

A B S T R A C TPresenilins (PS1 and PS2) exhibit similar γ-secretase-dependent and −independent functions with subtle variations. In this study, we established a cost-effective process to overexpress and purify full-length human PS2 in sufficient quantities and quality for structural studies. Upon optimization, milligram quantities of homogeneous trimeric hisPS2 were purified, which enabled the preliminary characterization of human hisPS2 zymogen. Far-UV and near-UV CD as well as fluorescence spectroscopy revealed that purified hisPS2 contained the expected secondary structure and was folded into a defined tertiary structure. Thermal stability analysis revealed a T m value of ∼55°C for secondary structure while cholesterol significantly increased the stability. The low melting temperature of ∼34°C for the tertiary structure was able to explain the purity and aggregation problems observed during purification. Additionally, the occurrence of calcium ions induced structural changes to different extents for PS2WT and PS2-D263A/D366A was observed, which is consistent with previous studies.

Data on solubilization, identification, and thermal stability of human Presenilin-2

“…To perform the detergent screening, equal amount of membrane suspensions (correspond to 0.1 g cell pellet) containing the expressed hisPS2 was extracted by 0.5 ml solubilization buffer containing the respective detergent ( Table 1 ). The detergent efficiency of extraction was classified by comparing the solubilization of hisPS2 in supernatant (solubilized fraction) and pellet (detergent resistant fraction) with the suspension before centrifugation at 100,000×g for 1 h ( Table 1 ) by western blot [1] . ( Table 2 ).…”

“…Chromatographically isolated trimeric full length hisPS2 [1] was analyzed on a 12% SDS-PAGE for his-tag positive western blot signals which were subjected to MS. Western positive bands were extracted and digested according to the Trypsin Profile IGD Kit (SIGMA) and acidified by addition of TFA to 0.1% final concentration. Sample of hisPS2 was analyzed by a Bruker Daltonic Ultraflex III TOF/TOF mass spectrometer using the software flex Analysis Version 3.0 (Bruker Daltonics, Build 92), and the peptides identified by searching against the protein database Swiss-Prot ( Fig.…”

“…CD spectra were measured using an Aviv CD-425 spectrometer from 260 to 185 nm for far UV and 350 to 250 nm for near UV [1] . The far UV CD spectra were deconvoluted by CDSSTR using the reference data sets 4, 7 and SMP 180 [6] .…”

“… The data presented here are related to the research article entitled “Expression, purification, and preliminary characterization of human presenilin-2" [1] . Human Presenilin-2 is the catalytic subunit of γ-secretase and a possible calcium leakage channel (Kimberly et al, 2000; Tu et al, 2006) [2] , [3] .…”

“…The data presented here are related to the research article entitled “Expression, purification, and preliminary characterization of human presenilin-2" [1] .…”