Fig 1.
Experimental set up and quantification scheme for Hsp90 overproduction.
(A) Schematic overview of the protein production experiment. E. coli cells containing human Hsp90β open reading frame were inoculated from glycerol stock and cultured overnight at 37°C. The culture was divided into small tubes and after induction the tubes were incubated in different conditions. Samples were taken at day 1, 3 and day 5 and were analysed by SDS-PAGE. (B) Schematic representation of the quantification of Hsp90 overproduction. The intensity profiles of uninduced (orange) and induced lanes (green) were determined and the induced lane profile were normalised to the uninduced lane profile using the EF-Tu peak of the uninduced lane as reference (100%) (top panel). Subsequently, the induced lane profiles were aligned to the uninduced lane profile using the first two left peaks (stars) of the uninduced lane profile as reference points (middle panel). The uninduced lane profile was deducted from the normalised and aligned induced lane profiles and the sum of difference at the height of the Hsp90 peak was calculated (red area) (lower panel). Finally, the Hsp90 peak area was divided by the normalised and aligned EF-Tu peak area.
Fig 2.
Hsp90 overproduction in LB medium lead to insufficient protein yield.
Hsp90 overproduction after 1 day (A), 3 days (B), 5 days (C) of induction. Lane 1: protein marker, lane 2: uninduced sample, lane 3–8: protein production at 16°C, lane 9–14: protein production at 18°C, lane 3–5 and 9–11: induction at low OD600, lane 6–8 and 12–14: induction at high OD600, lane 3, 6, 9, 12: induction with 0.1 mM IPTG, lane 4, 7, 10, 13: induction with 0.25 mM IPTG, lane 5, 8, 11, 14: induction with 0.5 mM IPTG, lane 15: purified Hsp90 sample. EF-Tu intrinsic E. coli protein band was used for quantification of Hsp90 overproduction.
Fig 3.
Hsp90 overproduction was improved but still insufficient in 2x YT compared to LB.
Hsp90 overproduction after 1 day (A), 3 days (B), 5 days (C) of induction. Lane 1: protein marker, lane 2: uninduced sample, lane 3–8: protein production at 16°C, lane 9–14: protein production at 18°C, lane 3–5 and 9–11: induction at low OD600, lane 6–8 and 12–14: induction at high OD600, lane 3, 6, 9, 12: induction with 0.1 mM IPTG, lane 4, 7, 10, 13: induction with 0.25 mM IPTG, lane 5, 8, 11, 14: induction with 0.5 mM IPTG, lane 15: purified Hsp90 sample. EF-Tu intrinsic E. coli protein band was used for quantification of Hsp90 overproduction.
Fig 4.
Hsp90 overproduction lead to the highest yields in TB medium.
Hsp90 overproduction after 1 day (A), 3 days (B), 5 days (C) of induction. Lane 1: protein marker, lane 2: uninduced sample, lane 3–8: protein production at 16°C, lane 9–14: protein production at 18°C, lane 3–5 and 9–11: induction at low OD600, lane 6–8 and 12–14: induction at high OD600, lane 3, 6, 9, 12: induction with 0.1 mM IPTG, lane 4, 7, 10, 13: induction with 0.25 mM IPTG, lane 5, 8, 11, 14: induction with 0.5 mM IPTG, lane 15: purified Hsp90 sample. EF-Tu intrinsic E. coli protein band was used for quantification of Hsp90 overproduction.
Fig 5.
Quantification of Hsp90 overproduction in different media.
(A) Summary of the quantification of Hsp90 overproduction in LB, after 1 day (white), 3 days (grey) and 5 days (black) of induction. Average of results of all conditions (1st column group), average of results on 16°C and 18°C (2nd and 3rd column groups), average of results at low or high OD600 at induction (4th and 5th column groups), average of results at 0.1, 0.25 and 0.5 mM IPTG concentration (6th to 8th column groups). The length of induction is the only variable that influences Hsp90 production. (B) Summary of the quantification of Hsp90 overproduction in 2x YT. The length of induction is the only variable that influences Hsp90 production. The yield is higher in 2x YT compared to LB. The structure of the chart is as in (A). (C) Summary of the quantification of Hsp90 overproduction in TB. The length of induction is the only variable that has a significant effect on Hsp90 production. The yield is higher in TB compared to LB and 2x YT. The structure of the chart is as in (A). The quantification is based on individual gels in Figs 2–4.
Fig 6.
The Hsp90 sample was free of degradation products and impurities after the three-step purification procedure.
(A) Schematic overview of the Hsp90 purification procedure. After cell disruption and centrifugation, the lysate was loaded first on a POROS 20MC Ni-column followed by a POROS 20HQ anion exchange column, then a heparin affinity chromatography column. Finally, the eluted protein sample was concentrated, the buffer was exchanged and the sample flash frozen. The time frame of the experiment is indicated on the bottom of the figure. The SDS-PAGE panels on the arrows show the purity of the samples after each purification step. (B) SDS-PAGE shows the purity of the Hsp90 sample after each purification step. Lane 1: peak fraction sample after POROS 20MC affinity chromatography purification step, lane 2: peak fraction sample after POROS 20HQ anion exchange purification step, lane 3: peak fraction sample after heparin affinity chromatography purification step.
Fig 7.
Purified Hsp90 sample incubated at physiological temperature for one day remained free of degradation products.
(A) SDS-PAGE about the stability of the purified Hsp90 sample. Protein marker (lane 1), Hsp90 incubated for 0 hours (lane 2), Hsp90 incubated at 4°C (lane 3–5), 21°C (lane 6–8), 37°C (lane 9–11), Hsp90 incubated for 3 hours (lane 3, 6, 9), 18 hours (lane 4, 7, 10), 24 hours (lane 5, 8, 11). (B) Silver staining about the stability of the purified Hsp90 sample. Protein marker (lane 1), Hsp90 incubated for 0 hours (lane 2), Hsp90 incubated at 4°C (lane 3–5), 21°C (lane 6–8), 37°C (lane 9–11), Hsp90 incubated for 3 hours (lane 3, 6, 9), 18 hours (lane 4, 7, 10), 24 hours (lane 5, 8, 11).
Fig 8.
Purified Hsp90 sample folded, sedimented correctly and its molecular mass was appropriate.
(A) CD spectrum of Hsp90 at 1.2 μM monomer concentration. The overall structure of the protein typically consists of α-helices. (B) The AUC spectrum of Hsp90 at 7.2 μM monomer concentration. Hsp90 dimers sediment with 5.6 S, whereas Hsp90 tetramers appear at 10.6 S. (C) SEC-MALLS spectra of the purified Hsp90 sample at 2.2 μM monomer concentration. The protein has the molecular weight of the dimer Hsp90 is 167.5 ± 13.3 kDa. The peak maximum is at 10.07 ml. Red line: light scattering, blue line: refractive index, green line: UV absorption at 220 nm, black dotted line: fit for molecular mass.