Authors: Robert Liddington & Andrey Bobkov
Introduction
Expression and purification of vinculin from bacterial culture.
Procedure
Starter Culture and Induction
- Pick a single colony (or from glycerol stock) into 25 ml TB + salts + 0.4% glucose + antibiotic
- Grow until mid-log phase (OD600 ~ 0.4-0.5), 37 °C
- Store at 4 °C O/N
- Inoculate 2×500 ml TB + salts + 0.4% glucose + antibiotic with 10 ml @ of starter culture
- Grow to OD600 = 0.4-0.6
- Induce with 1mM IPTG, 37 °C, 3 hours
- Pellet cells and resuspend in 20 ml of lysis buffer (300 mM NaCl, 10 mM Imidazole, 20 mM Tris pH 8)
- Quick freeze in Liquid Nitrogen. Store at -80 °C until needed
Purification
- Thaw Pellet(s) in room temperature water
- Add 2.5 ml 10 mg/ml lysozyme (10 mg/ml lysozyme in H2O stored at –20 °C) and 0.25 ml 100 mM PMSF (dissolve 100mM PMSF in EtOH and store at –20 °C) add βME to 5mM final to each pellet.
- Sonicate, large probe, 3-5 minutes, 1 second on 2 seconds off, on ice
- Spin 18 K, 1 HR in SS-34
- Wash HiTrap Chelating HP 5ml column (Amersham 17-0409-03) with 10 ml 6 M GuHCl (skip if a new column)
- Wash column with 50 ml dH2O
- Load column with 20 ml of 0.1 M NiCl2
- Wash column with 50 ml dH2O
- Wash column with 25 ml (5 column volumes) 10 mM Imadazole wash buffer (10 mM Imadazole, 500 mM NaCl, 20 mM Tris pH 8)
- Load sample containing 10 mM Imadazole, 300 mM NaCl, 20 mM TRIS pH 8, collect flow through. Check on gel.
- Wash column with 25 ml 10 mM Imadazole buffer (10 mM Imadazole, 500 mM NaCl, 20 mM Tris pH 8). Collect wash to check on gel.
- Wash column with 25 ml 25 mM Imadazole buffer (25 mM Imadazole, 500 mM NaCl, 20 mM Tris pH 8). Collect wash to check on gel.
- Wash column with 25 ml 75 mM Imadazole buffer (75 mM Imadazole, 500 mM NaCl, 20 mM Tris pH 8). Collect wash to check on gel.
- Elute sample with 12 ml 250 mM Imadazole buffer (250 mM Imadazole, 500 mM NaCl, 20 mM Tris pH 8). Check on gel
- Strip column with 10 ml EDTA strip buffer (50 mM EDTA, 500 mM NaCl, 20 mM Tris pH 8). Collect strip to check on gel.
- Rinse column with 50 ml dH2O, store at 4 °C
- Dialyze eluted, 12 ml sample against Buffer A (150 mM NaCl, 20 mM Tris pH 8, 5 mM EDTA, 5 mM βME) overnight at 4 °C.
- Dialyze 3 or more hours against Buffer B (150 mM NaCl, 20 mM Tris pH 8, 5 mM βME).
- Add 4 aliquots Thrombin (5 Units), 4 °C Overnight
- Add Imidazole to a final concentration of 250mM, NaCl to a final of 500mM.
- Repeat Nickel column Steps 5-18 (cleaved protein should be in the FT and Wash 1).
- Add another 2 aliquots of thrombin (2.5 U) to elution, RT overnight.
- Repeat Step 21.
- Concentrate to appropriate concentration and run over S-200 column.
Author information
Robert Liddington & Andrey Bobkov, The Burnham Institute
Source: Protocol Exchange (2009) doi:10.1038/nprot.2009.45. Originally published online 5 February 2009.