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

被引：1

作者：

Yang, Ge ^{[1
,2
]}

Yu, Kun ^{[1
,2
]}

Kubicek, Jan ^{[3
]}

Labahn, Joerg ^{[1
,2
]}

机构：

[1] CSSB FZJ, CSSB, Notkestr 85, D-22607 Hamburg, Germany

[2] Forschungszentrum Julich, Inst Complex Syst Struct Biochem ICS 6, Wilhelm Johnen Str, D-52425 Julich, Germany

[3] Cube Biotech GmbH, Alfred Nobel Str 10, D-40789 Monheim, Germany

来源：

PROCESS BIOCHEMISTRY | 2018年 / 64卷

关键词：

Presenilin-2; Expression; Purification; Secondary structure; Tertiary structure; Thermal stability; GAMMA-SECRETASE COMPLEX; MEMBRANE-PROTEIN OVEREXPRESSION; AMYLOID PRECURSOR PROTEIN; DISEASE-LINKED MUTATIONS; SINGLE-CHANNEL ACTIVITY; ALZHEIMERS-DISEASE; CALCIUM HOMEOSTASIS; HIPPOCAMPAL-NEURONS; RYANODINE RECEPTORS; CIRCULAR-DICHROISM;

D O I：

10.1016/j.procbio.2017.09.012

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Presenilins (PS1 and PS2) exhibit similar gamma-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 similar to 55 degrees C for secondary structure while cholesterol significantly increased the stability. The low melting temperature of similar to 34 degrees 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.

引用

页码：63 / 73

页数：11

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