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All proteins expressed in this thesis contained either a C- or an N-terminal FLAG- tag. The FLAG-tag itself is an octapeptide, which is made up of the amino acid sequence DYKDDDDK (Figure 17).

As for other tags used in molecular biology, the FLAG-tag with its rather unique peptide sequence, is used to affinity-purify only those proteins that contain a FLAG-tag and are over-expressed in Sf9 cells. For this, a commercially available resin that contains agarose-beads to which multiple copies of monoclonal anti- FLAG antibody are attached (Anti-FLAG M2® Affinity Gel, SIGMA) was used (Figure 18).

The size of the beads keeps the bound proteins from passing through the membrane of the column. Via competitive elution, where synthetic FLAG-Peptides are applied in excess amount to the solution of antibody-bound protein, the protein of interest is eventually eluted from the column. Statistically the antibody will bind more likely to the highly concentrated FLAG peptides than to the much lower concentrated proteins of interest (Figure 19). This way high yields of

relatively clean protein fractions are achieved.

Figure 17. Schematic of FLAG-affinity protein purification −

Part 1.

Depicted is the cell lysate fraction containing the overexpressed FLAG-tagged protein of interest (depicted is a double-headed motor protein, e.g. Myosin V).

Figure 18. Schematic of FLAG-affinity protein purification − Part 2.

Upon incubation with anti-FLAG® M2 agarose resin, FLAG-tagged proteins bind to the resin. Thereby highly efficient separation of the FLAG-tagged protein from other unwanted cytoplasmic protein fractions is achieved.

Figure 19. Schematic of FLAG-affinity protein purification − Part 3.

FLAG-tagged protein fraction is eluted from the column by competitive elution applying an excess amount of FLAG-peptides.

4.3.4.1 General FLAG-tag affinity purification protocol

All steps were carried out on ice at all times. Before harvesting of the cells, viability and density was determined for comparison of infection rates. The cells were harvested by centrifugation at 1,500 x g for 15 minutes at 6°C. The pelleted cells were resuspended in approx. 9% vol. (of the initial cell suspension volume) Lysis Buffer (LB) 1X and homogenized in a glass-bounce homogenizer for 5 minutes on ice. The homogenate was cleared by centrifugation at 69,000 × g for

15 minutes at 4°C (L8-70M Ultra-centrifuge and 42.1 Rotor, Beckman-Coulter). The supernatant, containing most of the soluble proteins, was mixed with 3% vol. (of lysis solution volume) anti-FLAG agarose resin (Anti-FLAG M2® Affinity Gel). This mixture of anti-FLAG resin and cellular proteins was then incubated rotating for 90 to 120 minutes at 4°C. As a next step, the mixture now containing the antibody-bound FLAG-tagged proteins of interest was applied onto a PolyPrep® chromatography column, which was equilibrated with 2 to 3 ml of LB 1X beforehand. The cushion of anti-FLAG resin on the column was washed 8 to 10 times with 2 ml of Wash Buffer (WB) 1X by constant stirring the suspension with a glass-stick. Protein elution from the column took place in 0.5 to 1.5 % vol. (of lysis suspension volume) of Elution Buffer (EB). Before the actual elution was carried out and under constant stirring, EB and the resin mixture were incubated for 30 minutes at 4°C (chromatography column sealed-up). By gently applying air pressure onto the unsealed column, protein elution was carried out. Immediately after elution, via Slide-A-Lyzer® dialysis cassettes (0.1 - 0.5 ml or 0.5 - 3 ml capacity, 10 kDa molecular weight cut-off) samples were dialyzed for 60 to 120 minutes at 4°C into 500 ml dialysis buffer (DB, containing 50% glycerol). This step was included to make the purified proteins suitable for long-term storage at -20°C. For SDS-PAGE analysis always 15 to 30 µl of flow-through (FT), wash (W), elution (E, prior dialysis) and dialysis (D) was saved. For those purifications where protein yield was expected to be low, protein concentration was further scaled up with Amicon Ultra-4 centrifugal filter units (10 kD molecular weight cut-off). For this, the filter unites containing the un-dialyzed protein elution, were centrifuged in a swinging bucket centrifuge (swinging-bucket centrifuge, Rotanta 460R, Hettich) at 4,000 x g for 5 to 10 minutes and 4°C.

for subsequent cryo-EM studies the CaMKII-α protein was dialyzed into 10%

glycerol (in DB 1X), keeping the protein stable at 4°C for four weeks. If snap- frozen in liquid nitrogen (for biochemical analysis) the protein could be stored at - 70°C for up to several months.

For protein-specific buffers used during cell lysis, sample washing and sample elution, please refer to Table 17.

HMM-like Myosin Va

FL-Xenopus

Myosin Va CaMKII-α

Lysis LB-HMM-M5 LB-XM5 Brickey Buffer

Wash WB-HMM-M5 WB-XM5 WB-CK

Elution EB-HMM-M5 EB-XM5 EB-CK

Dialysis _{DB-HMM-M5 DB-XM5 DB-CK}

4.3.4.2 Optimized protocol for FLAG-tag affinity purification of full-

length Xenopus l. Myosin Va

For the purification of full-length Xenopus l. Myosin Va, in principle the general FLAG-tag affinity purification protocol was followed (for details, refer to Section 4.3.4.1).

Due to strong degradation of the full-length protein, any detergent in the lysis buffer was omitted and instead of Dounce-homogenization, cell lysis was performed via sonication for a total of three minutes (duty cycle: 40%, output control: 4). Cell lysate was cleared at 27,000 × g (Sorvall RC-5B and SS-34 rotor)

for 30 minutes at 4°C. Because of its high molecular weight (larger than 215 kDa), the expression was expected to yield relatively low protein concentrations. Thus, immediately after the first incubation with anti-FLAG resin was completed the mixture was applied onto the column, and the flow-through of this first incubation round underwent a second round of incubation with anti-FLAG resin. Furthermore, two rounds of incubation with EB (1% total vol. of lysis solution volume) were performed while eluting the protein sequentially in-between.

Before dialysis into 50% glycerol, protein concentration was typically scaled-up

Table 17. Listing of the different buffers used during FLAG-tag affinity purification of the different baculovirus-expressed proteins of interest.

4.3.5 Methods for functional protein analysis