4 MATERIALS & METHODS 87
4.8 Protein expression and purification 96
4.8.1 Protein expression in E. coli
Colonies of freshly transformed BL21 Star (DE3) cells were used to inoculate 20 ml of LB supplemented with the respective antibiotics. After cultivation for 4 h at 37 °C, 3 l of prewarmed LB supplemented with the respective antibiotics (and with 2 g/l glucose, in case of maltose-‐binding protein (MBP)-‐fusion proteins) were inoculated with the preculture. At an OD600 of ca. 0.3, the culture was cooled to 18 °C over 30-‐60 min. At an OD600 of ca. 0.6, expression of the recombinant protein was induced by adding 0.25 mM Isopropyl-‐β-‐D-‐
thiogalactopyranosid (IPTG). The cells were cultured at 18 °C and 140 rpm for ca. 15-‐17 h. Afterwards, cells were pelleted (4400 x g, 15 min, 4°C), resuspended in the respective buffer, and either flash frozen with liquid nitrogen and stored at -‐80 °C or directly used for purification.
The expression of Myo4p (978-‐1471) was improved by using 2x TY medium supplemented with 100 µg/ml ampicillin. In addition, 2% (v/v) ethanol was added just before induction.
4.8.2 Protein expression in insect cells
Recombinant baculovirus was prepared essentially as described for the Bac-‐to-‐ Bac system (Invitrogen). In brief, 2 ml SF21 cells with a density of 0.4 x 106 cells/ml were transfected with 1-‐2 µg bacmid DNA using the FuGENE HD transfection reagent (Roche). After cultivation for at least four days at 27.5 °C, the supernatant (P0) of two transfections (4 ml) was used to infect 10 ml SF21 cells with a density of 1.4 x 106 cells/ml. After cultivation for four days at 27.5 °C and 95 rpm, the cells were centrifuged at 2000 x g, the supernatant (P1)
was sterile filtered, and 2 ml thereof were used to infect 500 ml SF21 with a density of 0.4 x 106 cells/ml. After cultivation for four days, cells were pelleted, the supernatant (P2) was sterile filtered and stored at 4 °C.
A culture of 500 ml High Five cells with a density of 1 x 106 cells/ml were infected with 10-‐30 ml P2 virus supernatant and cultivated for 60-‐70 h at 27.5 °C. Afterwards, the cells were collected by centrifugation (2000 x g, 15 min, 4 °C), resuspended in the respective buffer, and either flash frozen with liquid nitrogen and stored at -‐80 °C or directly used for purification.
4.8.3 Purification of She2p, She3p (1-234), and Myo4p (978-1471)
Wild-‐type and mutant She2p was purified essentially as described (Müller 2009), except that the glutathione S-‐transferase (GST)-‐tag was cleaved on the column. Purification of His-‐tagged She3p (1-‐234) and Myo4p (978-‐1471) was performed as previously described (Heuck 2009).
4.8.4 Purification of full-length She3p constructs
All steps were carried out at 4 °C. The cell pellet of a 0.5-‐1 l culture was resuspended in S3-‐Ni-‐A1 buffer to a total volume of ca. 40 ml. One tablet of ethylenediaminetetraacetic acid (EDTA)-‐free protease inhibitor cocktail (Roche) and 0.4 mM phenylmethylsulfonyl fluoride (PMSF) was added. The cell suspension was lysed by sonication (3x 2 min, amplitude: 35%, output: 6). The lysate was cleared by centrifugation (39000 x g, 2x 20 min), filtered with a 2.7 µM filter and loaded on a HisTrap nickel sepharose column (GE Healthcare). The column was washed successively with S3-‐Ni-‐A1 buffer, S3-‐Ni-‐A2 buffer, and S3-‐Hep-‐A buffer. Protein was eluted directly on a HiTrap Q sepharose column attached to a HiTrap heparin sepharose column (both: GE Healthcare) with 60% S3-‐Ni-‐B and 40% S3-‐Hep-‐A buffer. The heparin column was washed with S3-‐Hep-‐A buffer, 15% S3-‐Hep-‐B buffer, and bound protein was eluted with 35% S3-‐Hep-‐B buffer. The protein was concentrated by ultrafiltration with an Amicon
Ultra centrifugal filter (Millipore) and finally purified with a Superose 6 10/300 GL column (GE Healthcare) in S3-‐SEC buffer. Purified She3p was usually concentrated to 8-‐12 mg/ml by ultrafiltration. Aliquots were flash frozen in liquid nitrogen and stored at -‐80 °C. SDS-‐PAGE and UV-‐spectroscopy were used to analyze the quality of the purification. The ratio of absorbance at 260 nm and 280 nm was below 0.6 indicating that no contaminating nucleic acids were present.
S3-‐Ni-‐A1 buffer: 20 mM HEPES/NaOH (pH 7.8), 500 mM NaCl, 20 mM Imidazole S3-‐Ni-‐A2 buffer: 20 mM HEPES/NaOH (pH 7.8), 1 M NaCl, 40 mM Imidazole S3-‐Ni-‐B buffer: 20 mM HEPES/NaOH (pH 7.8), 200 mM NaCl, 500 mM Imidazole S3-‐Hep-‐A buffer: 20 mM HEPES/NaOH (pH 7.8), 200 mM NaCl
S3-‐Hep-‐B buffer: 20 mM HEPES/NaOH (pH 7.8), 1 M NaCl, 2 mM EDTA, 2 mM DTT S3-‐SEC buffer: 20 mM HEPES/NaOH (pH 7.8), 500 mM NaCl, 2 mM DTT
4.8.5 Purification of C-terminal She3p constructs
C-‐terminal She3p constructs were either purified via an N-‐terminal GST-‐ or MBP-‐ tag. In addition, a C-‐terminal 6x His-‐tag was present. All steps were carried out at 4 °C. The cell pellet was resuspended in S3C-‐GST-‐A1 buffer supplemented with EDTA-‐free protease inhibitor cocktail. The cell suspension was lysed by sonication (3x 4 min, amplitude: 35%, output: 6), the lysate was cleared by centrifugation (30000 x g, 30 min), and loaded on a GSTrap glutathione sepharose column (GE Healthcare). The column was washed successively with S3C-‐GST-‐A1 buffer, S3C-‐GST-‐A2 buffer, and again with S3C-‐GST-‐A1 buffer. A volume of 5 ml S3C-‐GST-‐A1 buffer supplemented with 100 µg PreScission protease (GE Healthcare) was applied to the column. After incubation at 4 °C for at least three hours, the cleaved protein was eluted directly on a HisTrap nickel sepharose column (GE Healthcare) with S3C-‐Ni-‐A buffer. The column was washed with 15% S3C-‐Ni-‐B buffer. Bound protein was eluted with S3C-‐Ni-‐B buffer, concentrated by ultrafiltration, and separated on a Superose 12 10/300 GL column (GE Healthcare) in S3C-‐SEC buffer. Pure fractions were pooled and concentrated by ultrafiltration. Since the She3p C-‐terminus shows no absorption at 280 nm, the concentration was estimated from sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-‐PAGE) with Coomassie blue staining. Aliquots were flash frozen with liquid nitrogen and stored at -‐80 °C.
In the case of MBP-‐tagged She3p variants, the cell pellet was resuspended in S3C-‐MBP-‐A buffer supplemented with 5 mM EDTA and protease inhibitor cocktail. The lysate was loaded on a gravity flow column filled with amylose resin (New England Biolabs). The column was washed with S3C-‐MBP-‐A buffer, bound protein was eluted with S3C-‐MBP-‐B buffer, and loaded on a HisTrap nickel sepharose column (GE Healthcare). The following steps of the purification were performed as described above.
S3C-‐GST-‐A1 buffer: 20 mM HEPES/NaOH (pH 7.8), 500 mM NaCl S3C-‐GST-‐A2 buffer: 20 mM HEPES/NaOH (pH 7.8), 1 M NaCl S3C-‐MBP-‐A buffer: 20 mM HEPES/NaOH (pH 7.8), 200 mM NaCl
S3C-‐MBP-‐B buffer: 20 mM HEPES/NaOH (pH 7.8), 200 mM NaCl, 10 mM Maltose S3C-‐Ni-‐A buffer: 20 mM HEPES/NaOH (pH 7.8), 500 mM NaCl, 20 mM Imidazole S3C-‐Ni-‐B buffer: 20 mM HEPES/NaOH (pH 7.8), 500 mM NaCl, 500 mM Imidazole S3C-‐SEC buffer: 20 mM HEPES/NaOH (pH 7.8), 200 mM NaCl
4.8.6 Purification of full-length Myo4p
All steps were carried out at 4 °C. The cell pellet of a 0.5-‐1 l culture was resuspended in M4-‐HB buffer to a total volume of ca. 40 ml and lysed by sonication (3x 2 min, amplitude: 35%, output: 6). The lysate was cleared by centrifugation (39000 x g, 2x 20 min), filtered with a 2.7 µM filter, and 1 ml ANTI-‐FLAG M2 affinity gel (Sigma-‐Aldrich) was added. The mixture was gently agitated for 60 min, transferred into a gravity flow column and washed with M4-‐FLAG-‐A buffer. Bound protein was eluted five times with 1 ml M4-‐FLAG-‐B buffer. The eluate was dialyzed two times against 1.5 l of ATPase buffer, followed by dialysis against 0.5 l ATPase buffer supplemented with 50% (v/v) glycerol. The protein was stored at -‐20 °C. The concentration of the protein was estimated by comparing the intensity of the Coomassie blue staining of Myo4p with known concentrations of bovine serum albumin (BSA) in an SDS-‐polyacrylmide gel.
M4-‐HB buffer: 20 mM Imidazole (pH 7.5), 200 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 7% (w/v) Sucrose, 1 mM PMSF, 3 mM ATP,
1 tablet EDTA-‐free protease inhibitor cocktail per 50 ml buffer M4-‐FLAG-‐A buffer: 20 mM Imidazole (pH 7.5), 150 mM KCl, 5 mM MgCl2, 1 mM EDTA,
1 mM EGTA, 0.5 mM PMSF, 3 mM ATP,
1 tablet EDTA-‐free protease inhibitor cocktail per 50 ml buffer M4-‐FLAG-‐B buffer: 20 mM Imidazole (pH 7.5), 150 mM KCl, 5 mM MgCl2, 1 mM EDTA,
1 mM EGTA, 0.5 mM PMSF, 0.1 mg/ml FLAG peptide,
1 tablet EDTA-‐free protease inhibitor cocktail per 50 ml buffer ATPase buffer: 20 mM HEPES/NaOH (pH 7.8), 100 mM NaCl, 25 mM KCl, 2 mM MgCl2,
1 mM EGTA, 2 mM DTT
4.8.7 Purification of actin from rabbit muscle
Actin was purified from 5 g rabbit muscle acetone powder (Sigma-‐Aldrich, M6890). All steps were carried out at 4 °C. The frozen acetone powder was crushed into small pieces and actin was extracted 6-‐8 times. Therefore, the powder was covered with G-‐buffer, stirred for 20-‐30 min, and filtered with a compress. The first filtrate was discarded. All other filtrates were pooled and cleared by ultracentrifugation (Type 45 Ti rotor, 41 krpm, 30 min). The supernatant was supplemented with 800 mM KCl, 2 mM MgCl2, and 1 mM ATP, the pH was adjusted to 8, and polymerization of actin was allowed to proceed over night under slow stirring. F-‐actin was pelleted by ultracentrifugation (Type 45 Ti rotor, 41 krpm, 3 h). The pellets were washed with G-‐buffer and resuspended in 3-‐5 ml of G-‐buffer with a dounce homogenizer. The solution was dialyzed two times against 2 l of G-‐buffer. After ultracentrifugation (Type 70.1 Ti rotor, 44 krpm, 2 h) the supernatant contained the finally purified G-‐actin. The concentration was determined by absorption at 290 nm using an extinction coefficient of 26000 M-‐1cm-‐1 (Abs 0.1% = 0.62). G-‐actin could be stored for up to two weeks at 4 °C when daily dialyzed against G-‐buffer supplemented with 0.02% (w/v) sodium azide. After polymerization (Section 4.11.15), F-‐actin could be flash frozen in liquid nitrogen and stored at -‐80 °C.
G-‐buffer: 2 mM TRIS/HCl (pH4°C 8), 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM DTT