Production, Purification and Crystallization of a Prokaryotic SLC26 Homolog for Structural Studies

Materials and Reagents

1. Dialysis tube
   MWCO 8 kDa (Carl Roth, catalog number: 1924.1 )
   MWCO 3.5 kDa (Carl Roth, catalog number: E860.1 )
   
2. Concentrators
   MWCO 50 kDa (EMD Millipore, catalog number: UFC905024 )
   MWCO 3 kDa (EMD Millipore, catalog number: UFC800324 )
   
3. Crystallization plates (HAMPTON RESEARCH, catalog number: HR3-158 )
   
4. Potter tube and piston (VWR, catalog numbers: 432-0205 and 432-0211 )
   
5. 50 ml tube
   
6. E. coli MC1061 (Coli Genetic Stock Center, catalog number: 6649 )
   
7. Plasmid pBXNPHM3-Nb5776 (Figure 1A)
   Note: This plasmid holds the gene coding for the nanobody under control of the araBAD promoter and results in the production of the nanobody fused to an N-terminal pelB leader sequence followed by decaHis-tag, MBP, and a HRV 3C protease site. It contains a beta-lactam antibiotic marker. Plasmid available for non-commercial use upon request.
   
8. Plasmid pBXC3GH-SLC26Dg (Figure 1B)
   Note: This plasmid holds the gene coding for SLC26Dg under control of the araBAD promoter and results in the production of SLC26Dg fused to a C-terminal HRV 3C protease site, GFP, and a decaHis-tag. It contains a beta-lactam antibiotic marker. Plasmid available for non-commercial use upon request.
   
   
   
   Figure 1. Plasmid maps. A. Plasmid map of pBXNPHM3-Nb5776; B. Plasmid map of pBXC3GH-SLC26Dg. Unique restriction sites and important features in the plasmids are indicated.
   
   
9. Ampicillin sodium salt (Carl Roth, catalog number: K029.2 )
   
10. L-arabinose (20% w/v in ddH₂O) (Carl Roth, catalog number: 5118.2 )
    
11. 1 M KPi, pH 7.5 (see Recipes)
    K₂HPO₄(AppliChem, catalog number: 121512 )
    KH₂PO₄(AppliChem, catalog number: 131509 )
    
12. Sodium chloride (NaCl, 2.5 M, ddH₂O) (Carl Roth, catalog number: 3957.1 )
    
13. Lysozyme (100 mg/ml in ddH₂O) (AppliChem, catalog number: A3711 )
    
14. DNAse I (2 mg/ml in ddH₂O) (AppliChem, catalog number: A3778 )
    
15. Magnesium sulphate heptahydrate (MgSO₄, 1 M, ddH₂O) (Carl Roth, catalog number: P027.2 )
    
16. Phenylmethyl sulphonyl fluoride (PMSF, 200 mM in ethanol) (Carl Roth, catalog number: 6367.1 )
    
17. NiNTA (50% w/v slurry in 20% EtOH) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 88223 )
    
18. ddH₂O
    
19. Imidazole (2 M, pH 7.5, HCl, ddH₂O) (Carl Roth, catalog number: X998.4 )
    
20. HEPES (1 M, pH 7.5, NaOH, ddH₂O) (Carl Roth, catalog number: HN78.2 )
    
21. HRV 3C protease (homemade)
    
22. Polypropylene glycol 2000 (10% v/v in ddH₂O) (Sigma-Aldrich, catalog number: 81380-1L )
    
23. EDTA, disodium salt dihydrate (Carl Roth, catalog number: X986.1 )
    
24. Glycerol, 86% (w/v) (Carl Roth, catalog number: 4043.3 )
    
25. Liquid nitrogen
    
26. Decylmaltoside (10% w/v in ddH₂O) (Anatrace, catalog numbers: D322S and D322LA )
    
27. Ammonium formate (Carl Roth, catalog number: 5093.1 )
    
28. Sodium acetate (Na-acetate) (Carl Roth, catalog number: 6773.2 )
    
29. PEG400 (Sigma-Aldrich, catalog number: 91893 )
    
30. Tryptone (AppliChem, catalog number: A1553 )
    
31. Yeast extract (AppliChem, catalog number: A1552 )
    
32. Agar (Applichem, catalog number: A0917 )
    
33. LB agar (see Recipes)
    
34. TB medium (see Recipes)
    
35. Reservoir solution (see Recipes)
    

Equipment

1. 5 L baffled flask
   
2. Incubator at 37 °C
   
3. Shaker with adjustable temperature (Infors, model: HT Multitron )
   
4. Centrifuge for pelleting cultures (Thermo Fisher Scientific, Thermo Scientific, model: Thermo Sorvall Evolution RC )
   
5. Homogenizer (IKA, model: ULTRA-TURRAX^® T25 )
   
6. High pressure cell disrupter (Avestin, model: Emulsiflex C3 )
   
7. Nanodrop (Thermo Fisher Scientific, Nanodrop^TM, model: 1000 , discontinued)
   
8. Magnet stirrer (Heidolph Instruments, model: Hei-Mix S )
   
9. SEC column Superdex 75 10/300 GL (GE Healthcare, catalog number: 17-5174-01 )
   
10. SEC column Superdex 200 10/300 GL (GE Healthcare, catalog number: 28-9909-44 )
    
11. Table-centrifuge (VWR, model: Micro Star 17 )
    
12. HPLC (GE Healthcare, model: ÄKTAprime plus )
    
13. Fermenter (BIOENGiNEERiNG, model: NLF 22, 30 L)
    
14. Spectrophotometer (Amersham Biosciences, model: Ultrospec 10 )
    Note: This product has been discontinued.
    
15. Ultracentrifuge (Beckman Coulter, model: Optima XPN-100K Ultracentrifuge )
    

Procedure