Expression, purification, and reconstitution of the transmembrane domain of the human amyloid precursor protein for NMR studies
Highlights
► APPTM has been successfully overexpressed as a fusion protein with MBP. ► Pure APPTM can be obtained by Ni-NTA chromatography, TEV protease cleavage and RP-HPLC. ► Isotopically labeled APPTM WT and an FAD mutant were purified for NMR studies. ► Excellent HSQC spectra were obtained in DPC and SDS micelles.
Introduction
Alzheimer’s disease (AD)2 is the most common type of dementia in elderly people [1]. One of the pathologic hallmarks of AD, senile plaque, is mainly composed of amyloid β peptide (Aβ). The length of the Aβ peptide usually ranges from 39 to 43 residues. The two major isoforms of Aβ in the brain are Aβ40 and Aβ42, consisting of 40 and 42 residues respectively [2], [3]. While Aβ40 is relatively benign, Aβ42 tends to aggregate rapidly into neurotoxic oligomers and fibrils (plaques) [4]. Aβ is produced from the amyloid precursor protein (APP) through two sequential proteolytic cleavages by β- and γ-secretases [5].
APP is a membrane protein with a single transmembrane (TM) domain (APPTM). The proteolytic cleavage of APP is initiated by β-secretase within the extracellular domain of APP, releasing a C-terminal membrane-anchored fragment of 99 residues (C99) [6], [7]. C99 is further cleaved by γ-secretase within the TM domain to yield Aβ and the APP intracellular domain (AICD) [8], [9], [10]. However, the cleavage of C99 by γ-secretase within the TM domain is not specific. Several cleavage sites have been identified, and Aβ peptides of varying length can be generated during γ-secretase cleavage. Aβ40 is predominantly released from the TM domain cleavage, and Aβ42 is generated to a lesser extent [4].
APPTM is the substrate of γ-secretase for the production of Aβ and the interaction between APPTM and γ-secretase is crucial for γ-secretase specificity, that is, the preference of Aβ40 production over Aβ42 production. Familial Alzheimer’s disease (FAD) is caused by missense mutations in genes encoding γ-secretase and APP. FAD mutations within APPTM alter γ-secretase specificity and lead to an increased Aβ42/Aβ40 ratio [11], [12]. Therefore, there is tremendous interest in obtaining the 3D structure of APPTM at atomic resolution and in understanding how the specifics of the structure may contribute to γ-secretase specificity.
Beel et al. [13] presented the first solution NMR studies of C99, which includes APPTM, and characterized the cholesterol binding properties of C99. However, due to the large size of the system (C99 + detergent micelle), complete side chain assignment, a prerequisite for NMR structure determination, was not achieved. Using a limited number of distance constraints from solid state NMR and molecular dynamics (MD) simulation, Sato et al. [14] obtained a structural model for APPTM. Computational models for APPTM have also been generated by Miyashita et al. [15], and recently Wang et al. [16]. To date, there is still no experimental 3D structure of APPTM at atomic resolution based on large numbers of distance and angular constraints.
To simplify the NMR spectra for structure determination, we have decided to pursue a smaller construct composed only of APPTM sequence, which was shown to be an excellent substrate for γ-secretase [17]. We started with APPTM with the V44M FAD mutation (V44 is named in according to Aβ numbering). We expressed recombinant APPTM V44M in Escherichia coli cells using the pETM41 vector (EMBL Protein Expression and Purification Facility). pETM41 uses maltose-binding protein (MBP) as the fusion partner which can increase expression level, stability and solubility. The His-tag at the N-terminus can be used for affinity purification. A TEV protease recognition site is present between MBP and APPTM, which is crucial for the release of APPTM from the MBP fusion protein. After successful expression using pETM41, we purified APPTM V44M and WT with isotopic labeling for NMR studies. Good HSQC spectra of APPTM WT and V44M have been obtained in DPC and SDS micelles.
Section snippets
Construction of APPTM expression vector
Codon optimized DNA of human APPTM mutant V44M was synthesized and cloned into the pZERO-2 vector by Integrated DNA Technologies (IDT). The DNA flanked with NcoI and BamHI restriction sites was digested and ligated into the NcoI and BamHI sites in the pETM41 vector, which encodes kanamycin resistance. After completion of cloning from pZERO-2 to pETM41, the DNA of pETM41-APPTM V44M was verified by DNA sequencing. The resulting plasmid encodes an N-terminally hexahistidine-tagged fusion protein
Overexpression and purification of APPTM
The expression protocol of recombinant APPTM is shown in Fig. 1A. The expression level of the MBP–APPTM fusion protein was induced by 1 mM IPTG followed by 24 h of growth at 30 °C (Fig. 1B). Clear differences can be observed on the SDS–PAGE gel between induced and uninduced cells. MBP–APPTM fusion protein (47 kDa) was the major product of the expression, accounting for about 70% of total protein, while it could not be detected without the addition of IPTG.
Purification procedures are also summarized
Discussion
Despite the importance of APPTM in the production of Aβ and the mechanism of Alzheimer’s disease, there is no experimental structure of APPTM at atomic resolution. NMR sample preparation is the first step for determining APPTM structure. Membrane proteins are inherently difficult to express and solubilize in aqueous solution due to their extremely hydrophobic nature. To resolve this problem, we used the MBP fusion to enhance the expression level and solubility of the expressed protein.
Acknowledgments
We are grateful for the financial support from American Health Assistance Foundation (A2009340). We thank European Molecular Biology Laboratory for providing the pETM41 vector.
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These two authors made equal contributions to this paper.