Expression and Purification of Chaperone-Active Recombinant Clusterin

<div><p>Clusterin was the first described secreted mammalian chaperone and is implicated as being a key player in both intra- and extracellular proteostasis. Its unique combination of structural features and biological chaperone activity has, however, previously made it very challenging to express and purify the protein in a correctly processed and chaperone-active form. While there are multiple reports in the literature describing the use of recombinant clusterin, all of these reports suffer from one or more of the following shortcomings: details of the methods used to produce the protein are poorly described, the product is incompletely (if at all) characterised, and purity (if shown) is in many cases inadequate. The current report provides the first well validated method to economically produce pure chaperone-active recombinant clusterin. The method was developed after trialling expression in cultured bacterial, yeast, insect and mammalian cells, and involves the expression of recombinant clusterin from stably transfected HEK293 cells in protein-free medium. The product is expressed at between 7.5 and 10 µg/ml of culture, and is readily purified by a combination of immunoaffinity, cation exchange and size exclusion chromatography. The purified product was shown to be glycosylated, correctly proteolytically cleaved into α- and β-subunits, and have chaperone activity similar to that of human plasma clusterin. This new method creates the opportunity to use mutagenesis and metabolic labelling approaches in future studies to delineate functionally important sites within clusterin, and also provides a theoretically unlimited supply of recombinant clusterin which may in the future find applications in the development of therapeutics.</p></div

Images of Coomassie blue stained SDS-PAGE gels analysing b-rCLU.

<p>(A) Lane 1: Molecular weight markers with size indicated in kDa. Lane 2: Sample of b-rCLU after SEC purification in solubilisation buffer containing 8 M urea and 5 mM DTT. (B) Lane 1: Molecular weight markers with size indicated in kDa. Lane 2: Sample of b-rCLU after step-wise dialysis into PBS/Az.</p

SEC and circular dichroism (CD) analyses of b-rCLU and wtCLU.

<p>(A) A₂₈₀ traces of SEC separations performed using a Superose™6 10/300 SEC column. The positions and masses (in kDa) of molecular weight markers are indicated by labelled arrows; the exclusion limit of the column (V_o) ≥4×10⁷ Da. For visual clarity the traces for b-rCLU (after step-wise dialysis into PBS) and wtCLU (in PBS) are deliberately vertically offset from that of b-rCLU fractionated in solubilisation buffer containing 8 M urea and 5 mM DTT. (B) CD plots represent mean molar ellipticity of 6 acquisitions. Similar results were obtained with IAA- and NEM-alkylated b-rCLU (data not shown).</p

Chaperone assays of m-rCLU and wtCLU using 3 client proteins.

<p>(A) BSA (1.5 mg/ml) was induced to precipitate using 20 mM DTT. The A₃₆₀ at 30 min intervals is plotted (mean ± standard deviation; n  = 3). (B) CS (200 µg/ml) and (C) CPK (1.12 mg/ml) were heated at 43°C to induce precipitation; the A₃₆₀ (mean ± standard deviation; n  = 3) is plotted at intervals of 5 min (CS) or 10 min (CPK). In all cases, m-rCLU and wtCLU were tested at a range of concentrations and representative plots shown, and OVA was used as a negative control (at the highest concentration of CLU tested or greater).</p

Data_Sheet_2_A Systematic Review of Commercial Cognitive Training Devices: Implications for Use in Sport.PDF

<p>Background: Cognitive training (CT) aims to develop a range of skills, like attention and decision-making, through targeted training of core cognitive functions. While CT can target context specific skills, like movement anticipation, much CT is domain general, focusing on core abilities (e.g., selective attention) for transfer to a range of real-world tasks, such as spotting opponents. Commercial CT (CCT) devices are highly appealing for athletes and coaches due to their ease of use and eye-catching marketing claims. The extent to which this training transfers to performance in the sporting arena is, however, unclear. Therefore, this paper sought to provide a systematic review of evidence for beneficial training effects of CCT devices and evaluate their application to sport.</p><p>Methods: An extensive search of electronic databases (PubMed, PsychInfo, GoogleScholar, and SportDiscus) was conducted to identify peer-reviewed evidence of training interventions with commercially available CT devices. Forty-three studies met the inclusion criteria and were retained for quality assessment and synthesis of results. Seventeen studies assessed transfer effects beyond laboratory cognitive tests, but only 1 directly assessed transfer to a sporting task.</p><p>Results: The review of evidence showed limited support for far transfer benefits from CCT devices to sporting tasks, mainly because studies did not target the sporting environment. Additionally, a number of methodological issues with the CCT literature were identified, including small sample sizes, lack of retention tests, and limited replication of findings by researchers independent of the commercial product. Therefore, evidence for sporting benefits is currently limited by the paucity of representative transfer tests and a focus on populations with health conditions.</p><p>Conclusions: Currently there is little direct evidence that the use of CCT devices can transfer to benefits for sporting performance. This conclusion, however, stems more from a lack of experimental studies in the sporting field and a lack of experimental rigor, rather than convincing null effects. Subsequently, there is an opportunity for researchers to develop more reliable findings in this area through systematic assessment in athletic populations and major methodological improvements.</p

Data_Sheet_1_A Systematic Review of Commercial Cognitive Training Devices: Implications for Use in Sport.PDF

<p>Background: Cognitive training (CT) aims to develop a range of skills, like attention and decision-making, through targeted training of core cognitive functions. While CT can target context specific skills, like movement anticipation, much CT is domain general, focusing on core abilities (e.g., selective attention) for transfer to a range of real-world tasks, such as spotting opponents. Commercial CT (CCT) devices are highly appealing for athletes and coaches due to their ease of use and eye-catching marketing claims. The extent to which this training transfers to performance in the sporting arena is, however, unclear. Therefore, this paper sought to provide a systematic review of evidence for beneficial training effects of CCT devices and evaluate their application to sport.</p><p>Methods: An extensive search of electronic databases (PubMed, PsychInfo, GoogleScholar, and SportDiscus) was conducted to identify peer-reviewed evidence of training interventions with commercially available CT devices. Forty-three studies met the inclusion criteria and were retained for quality assessment and synthesis of results. Seventeen studies assessed transfer effects beyond laboratory cognitive tests, but only 1 directly assessed transfer to a sporting task.</p><p>Results: The review of evidence showed limited support for far transfer benefits from CCT devices to sporting tasks, mainly because studies did not target the sporting environment. Additionally, a number of methodological issues with the CCT literature were identified, including small sample sizes, lack of retention tests, and limited replication of findings by researchers independent of the commercial product. Therefore, evidence for sporting benefits is currently limited by the paucity of representative transfer tests and a focus on populations with health conditions.</p><p>Conclusions: Currently there is little direct evidence that the use of CCT devices can transfer to benefits for sporting performance. This conclusion, however, stems more from a lack of experimental studies in the sporting field and a lack of experimental rigor, rather than convincing null effects. Subsequently, there is an opportunity for researchers to develop more reliable findings in this area through systematic assessment in athletic populations and major methodological improvements.</p

Well-Tempered Metadynamics as a Tool for Characterizing Multi-Component, Crystalline Molecular Machines

The well-tempered, smoothly converging form of the metadynamics algorithm has been implemented in classical molecular dynamics simulations and used to obtain an estimate of the free energy surface explored by the molecular rotations in the plastic crystal, octafluoronaphthalene. The biased simulations explore the full energy surface extremely efficiently, more than 4 orders of magnitude faster than unbiased molecular dynamics runs. The metadynamics collective variables used have also been expanded to include the simultaneous orientations of three neighboring octafluoronaphthalene molecules. Analysis of the resultant three-dimensional free energy surface, which is sampled to a very high degree despite its significant complexity, demonstrates that there are strong correlations between the molecular orientations. Although this correlated motion is of limited applicability in terms of exploiting dynamical motion in octafluoronaphthalene, the approach used is extremely well suited to the investigation of the function of crystalline molecular machines

Examples of conformational dependent α₂M activities.

<p>* α₂M dimers generated by hypochlorite treatment.</p><p>† α₂M dimers generated by thiocyanate treatment.</p><p>‡ α₂M dimers generated by SDS treatment.</p><p>Many of the activities of α₂M are dependent on whether or not the protein is in its native conformation or in its transformed state. Additionally, dissociation of the native α₂M tetramer into dimers (that can be induced using several different chemical methods) has also been demonstrated to influence the activities of α₂M.</p

The effect of lyophilization from Tris buffer on purified α₂M.

<p>Images of native PAGE (3–8% Tris-acetate) gels showing the migration of (A) reconstituted α_<b>2</b>M that had been lyophilized from 20 mM Tris, 130 mM glycine, 80 mM trehalose, pH 8.0 and (B) α_<b>2</b>M stored in solution at 4°C in 20 mM Tris, pH 8.0 for 2 months or following reconstitution after it had been lyophilized from 20 mM Tris, pH 8.0 and stored at -20°C for 7 days. As references, the positions of native and transformed α_<b>2</b>M are also shown in (A). (C) Matched α_<b>2</b>M samples in 20 mM phosphate, pH 7.4 or 20 mM Tris, pH 8.0 stored at 4°C for 2 months. Both samples contained 0.02% (w/v) sodium azide.</p

The effect of storage at -20°C on α₂M chaperone activity.

<p>(A) Images of native PAGE (3–8% Tris-acetate) analyses of α_<b>2</b>M stored in 20 mM phosphate buffer, pH 7.4 (4°C or -20°C, 1 month). The latter sample was rapidly frozen in LN prior to storage at -20°C. (B) Corresponding bisANS fluorescence measurements for the α_<b>2</b>M samples described in (A). The results shown are the values of the mean bisANS fluorescence (n = 3±SD) in AFU. * Denotes significantly increased bisANS fluorescence as a result of storage at -20°C (Student’s t-test p < 0.01) (C) Turbidity measurements of CPK aggregation in the presence or absence of α_<b>2</b>M which had been stored at 4°C or -20°C as described in (A). The data are from individual measurements and are representative of several different experiments.</p