Purification of Hsp90, Hsp90 Variants and Co-Chaperones

For protein purification, the cell suspension was thawed and DNase was added. Cell disruption was performed with a french press at 1.8 kbar. To separate cell fragments from soluble proteins, the cell lysate was centrifuged at high speed (45 min, 18000 rpm, 6°C).

All buffers were freshly prepared, filtered and degased prior use. As required, 1 mM DTT was added prior to use.

111 Figure 51: Overview of different purification strategies of several Hsp90s and co-chaperones.

112

113

The purification of Hsp90 wt, Hsp90 variants and co-chaperones from E.coli lysate was achieved by several chromatography techniques depending on the used tag, cleavage site, molecular weight or chemical parameters like isoelectric point (pI) of the protein (Figure 51).

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Affinity Chromatography

As all used constructs were N-terminally tagged with 6 histidines (6xHis), the initial step of the purification procedure was an affinity chromatography performed with a 5 ml packed HisTrap FF (GE Healthcare) column. The column was equilibrated, cleaned and stored according to the manufacturer’s instructions (GE Healthcare). The histidine-tagged proteins were immobilized by nickel ions affinity chromatography and separated from other proteins in the lysate. To ensure removing of unspecific bound proteins to the column material, a washing step with 30 column volume of 6 % buffer B was performed. The histidine-tagged proteins were eluted by setting up a step-gradient with 10 column volume to 60 % of buffer B. The increase in imidazole competes with the histidine-tagged protein for the binding site to the nickel ions and thereby displaces the protein.

The elution was collected in 5 ml fractions (note: 1 mM EDTA was added prior use to the tubes) and the fractions containing protein were detected by UV absorption at 280 nm. The column was finally washed with 10 column volume of 100 % of buffer B. The fractions containing the protein were analyzed by SDS-PAGE, then pooled and diluted in ResoureceQ buffer A to a volume of max. 150 ml.

Ion-Exchange Chromatography

Next, the protein was further purified by exchange chromatography. The basic principle of exchange chromatography is the reversible adsorption of macromolecules to the immobilized ion-exchange groups of opposite charge. For the purification of Hsp90, a ResourceQ (6 ml) column was applied. The column was equilibrated, cleaned and stored according to manufacturer’s instructions (GE Healthcare). The protein was loaded on the pre-equilibrated column and washed with 10 column volumes at a flow of 4 ml/min. In a fast first step, the salt gradient was increased from 0 mM to 100 mM KCl within 10 ml, followed by a slow linear gradient by increasing the salt concentration from 100 mM KCl to 500 mM KCl within 150 ml. Here, the elution of the protein from the column takes place. Finally, the column was washed by 100 % ResourceQ buffer B. Samples were taken from the collected fractions (5 ml) containing protein and were verified via SDS-PAGE analysis. The protein containing fractions were pooled and dialyzed in a dialysis tube (Spectra/Por®; molecular porous membrane tubing; Spectrum laboratories Inc., MWCO 6-8 kDa) twice for 1h against HAT buffer A at 4 °C.

115 Hydroxyapatite Chromatography

The hydroxyapatite chromatography is a “mixed mode” ion exchange chromatography since both anionic (Ca²⁺, NH4+

) and cationic groups (PO4

3-, COO^-) are involved (Bernardi, 1973). The HAT column (CV 30 ml) was equilibrated with 5 column volumes of HAT buffer A. The protein was loaded onto the column and washed with 5 column volumes of HAT buffer A. Next, a linear gradient from 0 mM KP to 400 mM within 200 ml was set up to ensure separation and elution of the proteins. The protein containing fractions were analysed, pooled and concentrated by centrifugation (3800 rpm, 20 min, 4 °C) with an Amicon filter unit up to a final volume of 5 ml.

Size-Exclusion-Chromatography

Finally, size-exclusion chromatography (SEC) or also called gel-filtration chromatography was used as the last purification step for all proteins. The principle of SEC is to separate proteins in solution according to their size and shape. Small macromolecules retain longer in the matrix pores and elute later compared to larger ones. The SEC column was equilibrated, cleaned and stored according to the manufacturer’s protocol (Pharmacia Biotech). The column size was chosen according the size of the protein: for Hsp90 and Sti1: HiLoad 16/60 Superdex 200 prep grade; Pharmacia Biotech// for Aha1:

HiLoad 16/60 Superdex 75 prep grade; Pharmacia Biotech). The concentrated protein solution was injected and the column was washed with 1 column volume of SEC buffer. The fraction size was 5 ml per fraction and the flow rate 1 ml/min. The protein-containing fractions were analysed, pooled and concentrated by centrifugation (3800 rpm, 20 min, 4 °C) with an Amicon filter unit (Millipore) up to a final concentration of about 100 µM. The pure protein was aliquoted, shock frozen in liquid nitrogen and kept at -80 °C until further use.

Dialysis

To exchange buffers, the protein was dialyzed against the respective buffer in a dialysis tube (Spectra/Por®; molecular porous membrane tubing; Spectrum laboratories Inc., MWCO 6-8 kDa)over night at 4 °C. For smaller volumes (100 - 500 µl) a Dialysis Slide-A-Lyzer™ cassette was used.

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Proteolytic Digest with SUMO Protease

In the case of N-terminal 6xHis-SUMO-tagged Hsp90, the SUMO-tag was removed after the ion-exchange chromatography. The Ubiquitin-like protease (ULP1/SUMO protease) hydrolyses a peptide bond in the sequence Gly-Gly-|-Ala-Thr-Tyr at the C-terminal end of the small ubiquitin-like modifier (SUMO) peptide (Li and Hochstrasser, 1999). To this end, 100 µl yeast SUMO protease (Ulp1) was added to the pooled fractions after IEX and at the same time the protein was dialyzed against Ni NTA buffer A over night. After cleavage, the SUMOtag and the protease were removed by a subsequent affinity chromatography. The untagged Hsp90 was collected, concentrated by centrifugation (3800 rpm, 20 min, 4 °C) with an Amicon filter unit up to a final volume of 5 ml and applied to a size-exclusion column.