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. 2022 Jan 10;27(2):418.
doi: 10.3390/molecules27020418.

High-Efficiency Expression and Purification of DNAJB6b Based on the pH-Modulation of Solubility and Denaturant-Modulation of Size

Sara Linse  1
Affiliations

High-Efficiency Expression and Purification of DNAJB6b Based on the pH-Modulation of Solubility and Denaturant-Modulation of Size

Sara Linse. Molecules. .

Abstract

The chaperone DNAJB6b delays amyloid formation by suppressing the nucleation of amyloid fibrils and increases the solubility of amyloid-prone proteins. These dual effects on kinetics and equilibrium are related to the unusually high chemical potential of DNAJB6b in solution. As a consequence, the chaperone alone forms highly polydisperse oligomers, whereas in a mixture with an amyloid-forming protein or peptide it may form co-aggregates to gain a reduced chemical potential, thus enabling the amyloid peptide to increase its chemical potential leading to enhanced solubility of the peptide. Understanding such action at the level of molecular driving forces and detailed structures requires access to highly pure and sequence homogeneous DNAJB6b with no sequence extension. We therefore outline here an expression and purification protocol of the protein "as is" with no tags leading to very high levels of pure protein based on its physicochemical properties, including size and charge. The versatility of the protocol is demonstrated through the expression of an isotope labelled protein and seven variants, and the purification of three of these. The activity of the protein is bench-marked using aggregation assays. Two of the variants are used to produce a palette of fluorescent DNAJB6b labelled at an engineered N- or C-terminal cysteine.

Keywords: extraction; self-assembly; solubilization.

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Conflict of interest statement

The author declares no conflict of interest.

Figures

Figure 1
Figure 1
Expression of DNAJB6b wt and mutants. (A) Sequence of DNAJB6b wt and the substitutions made in the mutants of this study. (B) Prediction of DNAJB6b wt using Alpha-fold2 [28] with the J-domain shown in red, the C-terminal domain in blue, Pro96 in yellow, Ser190, Ser192, Thr193, Ser194 and Thr195 in pink. (C) Whole E. coli extract after expression of DNAJB6b wt and mutants in rich medium (lane 1, 4, 5, 7, 9, 11, 13 and 15) or M9 minimal medium (lane 2). Lane 1, 2 = wt DNAJB6b. Lane 3 = Mw standard with the Mw of the 7 smallest proteins given to the left of lane 1. Lane 4 = NCys- DNAJB6b. Lane 5 = CCys- DNAJB6b. Lane 7 = DNAJB6b-ST5A. Lane 9 = DNAJB6b -ST18A. Lane 11 = DNAJB6b-ΔST. Lane 13 = DNAJB6b -P96R. Lane 13 = DNAJB6b -T193A. Cell pellets from 1 mL were dissolved in 800 µL of 8 M urea, pH 8.0, mixed 1:1 with SDS loading buffer and 3 µL loaded per lane. The quantity loaded in each lane thus corresponds to cells from 2 µL culture.
Figure 2
Figure 2
Initial isolation of DNAJB6b from E. coli cell pellet. (A) Outline of the isolation steps. 1–5. Sonication in 20 mM MES, pH 6.0 (supernatant after centrifugation). 6. Sonication in 10 mM Tris/HCL, 1 mM EDTA, pH 8.0. 7. Passage through Q-sepharose big beads. 8. Passage through SP sepharose HP. (B) SDS PAGE on a 10–20% polyacrylamide gel with lane 1–5 loaded with sonicate 1–5, lane S with Mw standard with sizes given to the right of the gel, lane 6 with sonicate 6, lane 7 with the Q flow-through, and lane 8 with the SP sepharose flow-through. The total time required for step 1–8 is ca. 2 h. The flow-through of SP sepharose (lane 8) was used for purification of the protein using ammonium sulphate precipitation and size-exclusion chromatography (see Figure 3).
Figure 3
Figure 3
Purification of DNAJB6b using size exclusion chromatograph. (A) An aliquot from the flow-through of SP sepharose (see lane 8 in Figure 2) was precipitated by AMS and the 10–21% fraction dissolved in 10 mL 6 M GuHCl, 20 mM sodium phosphate, 0.2 mM EDTA, pH 8.0 and injected on a 26/600 Superdex200 column operated in 2 M GuHCl, 20 mM sodium phosphate, 0.2 mM EDTA, pH 8.0. (B) SDS PAGE of fractions 9–14 on a 10–20% polyacrylamide gel. Fraction 10–13 were concentrated and injected on a 16/600 Superose6 column. (C) The elution of the 16/600 Superos6 column operated in 20 mM sodium phosphate, 0.2 mM EDTA, pH 8.0. The injected sample was 5 mL of fractions 10–13 from panels A, B lyophilized down to 1/3 of the original volume, i.e., in 6 M GuHCl, 60 mM sodium phosphate, 0.6 mM EDTA, pH 8.0. (D) SDS PAGE of fractions 8–21 on a 10–20% polyacrylamide gel. In panels A and C, the absorbance at 280 and 214 nm are shown in blue and purple, respectively. Fractions 11–18 are kept for use in biophysical experiments.
Figure 4
Figure 4
Concentration determination. (A) Absorbance spectra of fractions 13–16 from the elution of Superos6 as shown in Figure 3C,D. A small sample from each fraction was mixed 3:1 with 8 M GuHCl to bring the samples to 2 M GuHCl, which leads to the dissociation of the large polydisperse oligomers. (B) Absorbance spectrum of fraction 14 in buffer (pink), in which case the large polydisperse oligomers lead to significant light-scattering prohibiting concentration determination, and in 2 M GuHCl, which permits absorbance to be used for concentration determination.
Figure 5
Figure 5
Fluorescent DNAJB6b. (A,B) Examples of SDS PAGE after labelling and SEC to remove free dye for DNAJB6b-CCys labelled with Alexa-488. (A)The gel imaged on a “dark reader” with blue excitation filter and orange emission filters. (B) The same gel photographed after staining with coomassie (quick stain). Panel (C) shows fluorescence emission spectra recorded for samples with 1 µM constant concentration of DNAJB6b-CCys-Alexa-488 and varying concentrations of DNAJB6b-CCys-Alexa-555: 0 (black), 0.25 (marine), 0.5 (blue), 0.75 (light blue), 1.0 (cyan), 1.25 (green), 1.5 (yellow), 1.75 (orange) and 2.0 µM (red). (D) Native gel electrophoresis. Agarose gel imaged on an IR fluorescence scanner. In each well is loaded 5 nM DNAJB6b-CCys-IR680 mixed with different concentrations of unlabeled DNAJB6b-wt to yield the following total concentrations of DNAJB6b: 5 nM (lane 1), 9 nM (2), 13 nM (3), 21 nM (4), 37 nM (5), 69 nM (6), 133 nM (7), 255 nM (8), 505 nM (9), 1.0 µM (10), 2.0 µM (11), 4.0 µM (12), 8.0 µM (13), 16.0 µM (14), 32 µM (15). The gel is oriented with the positive pole at the top of the image and the negative pole at the bottom.
Figure 6
Figure 6
Aggregation kinetics. The activity of the purified proteins was validated by monitoring the fibril formation of 4 µM Aβ42 by ThT fluorescence in the absence (black) and presence (colors) of DNAJB6b-wt (A) or DNAJB6b-STA5 (B) at concentrations ranging from 2 to 150 nM, i.e., at molar ratios ranging from 0.0005 to 0.0375. The solid lines show fitted curves assuming inhibition of primary nucleation. The half times of aggregation as extracted from the data in panels A and B are shown as averages and standard deviations over 4 replicates for DNAJB6b-wt (black) and DNAJB6b-STA5 (red) with linear (C) and logarithmic (D) axes.
Figure 7
Figure 7
Net charge of DNAJB6b (A) and its parts (B) as a function of pH titration as calculated based on model compound pKa values [29].
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