
Figure 1. Coomassie stained SDS-PAGE of purified, heterologously expressed proteins from C. reinhardtii. M: MW marker PageRuler Prestained Protein Ladder 10-170 kDa; a) purified PFR1 loaded onto a 10% SDS-polyacrylamidgel; b) purified [2Fe2S] ferredoxin (PetF) loaded onto a 15% SDS-polyacrylamidgel; c) purifiedhydrogenase (HydA1) loaded onto a 10% SDS-polyacrylamidgel. Different amounts of protein are loaded onto each gel.
Materials and Reagents
- Expression vector (pASK-IBA)
- LB medium (Lennox) (Carl Roth)
- Vogel Bonner minimal medium (homemade) (Vogel HJ et al., 1956)
- Thiamin hydrochlorid (Carl Roth)
- Resazurin (Riedel-de Haën)
- Immidazole (Alfa Aesar)
- Escherichia coli BL21 (DE3) ΔiscR
- Clostridium acetobutylicum ATCC 824
- Ni Sepharose 6 Fast Flow (GE Healthcare)
- Strep-Tactin Superflow (IBA Gmb)
- Ampicillin
- Anhydrotetracycline
- Glucose
- Sodium dithionite (laboratory reagent grade > 85%)
- Avidin (Affiland)
- Strep tactin
- Glycerol
- d-desthiobiotin (≥ 98%, TLC)
- Thiamine pyrophosphate
- PageRuler Prestained Protein Ladder 10-170 kDa (Thermo Fisher Scientific, catalog number: 26616 )
- 0.1 M Tris buffer (pH 8) (see Recipes)
- Pre-equilibrated gravity flow Ni-NTA (see Recipes)
Equipment
- Airtight vial
- Sonicator: Branson Sonifier 250 (Branson)
- Ultracentrifuge
- Anaerobic tent (1% H2, 99% N2) (Toepffer Lab Systems)
- 0.2 μm pore size sterile filter (Sarstedt AG & Co.)
- NanoDrop (Paqlab, Germany)
- Batch fermenter (Infors HT, CH)
Procedure
- Anaerobic expression of pyruvate: ferredoxin oxidoreductase (Noth et al., 2013)
- Electroporation (Sambrook et al., 2006) of 100 μl E. coli BL21 (DE3) ΔiscR (Akhtar et al., 2008) with ~100 ng expression vector (pASK-IBA).
- Inoculation of 200 ml LB and aerobic growth of a preculture over night at 37 °C (180 rpm).
- Inoculation of 4 L Vogel Bonner medium (8 x 500 ml; 2,000 ml Erlenmeyer flasks) supplemented with 100 μg/ml ampicillin, 50 μM thiamin hydrochlorid and 0.2 μM resazurin using 15 ml preculture each.
- Aerobic growth at 37 °C and 180 rpm until the culture reaches the anaerobic phase at A600 of 0.6. At that point, the redox indicator resazurin within the medium turns from blue to pink.
- Each 2 L of culture are induced by adding 0.2 μg/ml anhydrotetracycline and transferred into sterile 2 L Schott flasks containing 50 ml 20% glucose (5 g/L).
- Protein expression is carried out over night at 8 °C without stirring.
- Cells are anaerobically harvested by centrifugation for 20 min at 7,500 x g, resuspended in Tris-HCl (pH 8.0), 10% glycerol and stored at -20 °C until purification.
- Anaerobic purification of pyruvate: ferredoxin oxidoreductase (His-tag)
- For purification the pellet (2 L of culture) is thawed at room temperature and lysed by sonication while keeping the cells cooled on ice.
Note: Five times for 30 sec; output, 25; Branson Sonifier 250.
- Sedimentation of cell debris at 200,000 x g for 60 min and 4 °C in an ultracentrifuge.
- The soluble fraction is filtered using a pore size of 0.2 μm to get rid of unwanted material which clogs the column.
- Then, the sample is loaded on a pre-equilibrated (100 mM Tris-HCl, pH 8.0, 10 mM imidazole, 0.5 mM thiamine pyrophosphate) gravity flow Ni-NTA fast-flow column with a bed volume of 4 ml.
- Protein purification is achieved via increasing the imidazole concentration from 10 to 20 mM during washing each with 40 ml buffer.
- The His-tagged PFR1 protein is eluted from the column with 10 ml buffer containing 100 mM imidazole. Nine elution fractions each 1.1 ml are collected.
- The protein concentration of the brownish main elution fractions 3 and 4 are immediately determined using A280.
- Aerobic expression of [2Fe2S] ferredoxins (Jacobs et al., 2009; Winkler et al., 2009) with minor changes
- E. coli BL21 (DE3) ΔiscR containing the expression plasmid pASK-IBA7-FDX is grown in Vogel Bonner minimal medium for 4 h after induction at A600 of 0.6.
- Cells are harvested, washed in Tris-HCl (pH 8.0), sedimented again and stored at -20 °C until purification.
- Anaerobic expression of HydA1 (Girbal et al., 2005; von Abendroth et al., 2008)
- Expression plasmid containing C. acetobutylicum ATCC 824 strain is grown in CGM-medium and a glucose concentration of 60 g/L anaerobically in a batch fermenter over night at 35-37 °C and 100 rpm.
- Cells are harvested in an anaerobic tent analog to E. coli, resuspended in Tris-HCl (pH 8.0), 10% glycerol containing 10 mM sodium dithionite and stored at -20 °C until purification.
- Anaerobic expression of bacterial 2[4Fe4S] ferredoxin analog to HydA1 (Girbal et al., 2005; von Abendroth et al., 2008, Noth et al., 2013)
- Expression plasmid containing C. acetobutylicum ATCC 824 strain is grown in CGM-medium and a glucose concentration of 60 g/L anaerobically in a batch fermenter over night at 35-37 °C and 100 rpm.
- Cells are harvested in an anaerobic tent analog to E. coli, resuspended in Tris-HCl (pH 8.0), 10% glycerol containing 10 mM sodium dithionite and stored at -20 °C until purification.
- Anaerobic purification of StrepII-tagged proteins (C-E)
- All buffers used contain 2 mM sodium dithionite.
- For purification the cell pellet is thawed at room temperature and lysed by sonication while keeping the cells cooled on ice.
Note: Five times for 30 sec; output, 25; Branson Sonifier 250.
- Sedimentation of cell debris at 200,000 x g for 60 min and 4 °C in an ultracentrifuge.
- Supernatant (40 ml) is incubated for 1 hour with 3.5 mg Avidin (Stock 50 mg.ml-1) at 4 °C.
- The soluble fraction is filtered using a poresize of 0.2 μm to get rid of biotinylated, complexed proteins and unwanted material which clogs the column.
- Then, the filtered solution is loaded on a Tris-HCl (pH 8.0) equilibrated 2 ml strep tactin gravity flow column.
- The unbound proteins are washed from the column using 80 ml Tris-HCl (pH 8.0).
- Elution is performed with 10 ml Tris-HCl (pH 8.0), d-desthiobiotin (0.8 mg/ml) in fractions of 1 ml.
Recipes
- 0.1 M Tris buffer (pH 8) (1,000 ml)
Mix 12.114 g of Tris base with 800 ml dH2O
Add 100 ml Glycerol
pH to 8 with HCl
Add ddH2O to 1,000 ml
Autoclave for 20 min at 121 °C
Store at 4 °C
- Pre-equilibrated gravity flow Ni-NTA
0.1 mM Tris-HCl (pH 8)
10 mM imidazole
0.5 mM thiamine pyrophosphate
Acknowledgments
Aerobic expression of [2Fe2S] ferredoxins was adapted from Jacobs et al. (2009). Anaerobic expression and purification of the 2[4Fe4S] bacterial type ferredoxin was done according to the previously published isolation of [FeFe]-Hydrogenase HydA1 from Chlamydomonas reinhardtii by Girbal et al. (2005) and von Abendroth et al. (2008), which is also presented here. Research on the pyruvate:ferredoxin oxidoreductase from C. reinhardtii was scientifically supported by Anja Hemschemeier and Thomas Happe.
References
- Noth, J., Krawietz, D., Hemschemeier, A. and Happe, T. (2013). Pyruvate:ferredoxin oxidoreductase is coupled to light-independent hydrogen production in Chlamydomonas reinhardtii. J Biol Chem 288(6): 4368-4377.
- Akhtar, M. K. and Jones, P. R. (2008). Deletion of iscR stimulates recombinant clostridial Fe-Fe hydrogenase activity and H2-accumulation in Escherichia coli BL21(DE3). Appl Microbiol Biotechnol 78(5): 853-862.
- Girbal, L., von Abendroth, G., Winkler, M., Benton, P. M., Meynial-Salles, I., Croux, C., Peters, J. W., Happe, T. and Soucaille, P. (2005). Homologous and heterologous overexpression in Clostridium acetobutylicum and characterization of purified clostridial and algal Fe-only hydrogenases with high specific activities. Appl Environ Microbiol 71(5): 2777-2781.
- Jacobs, J., Pudollek, S., Hemschemeier, A. and Happe, T. (2009). A novel, anaerobically induced ferredoxin in Chlamydomonas reinhardtii. FEBS Lett 583(2): 325-329.
- Sambrook, J. and Russell, D. W. (2006). Transformation of E. coli by Electroporation. CSH Protoc 2006(1).
- Vogel, H. J. and Bonner, D. M. (1956). Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem 218(1): 97-106.
- von Abendroth, G., Stripp, S., Silakov, A., Croux, C., Soucaille, P., Girbal, L. and Happe, T. (2008). Optimized over-expression of [FeFe] hydrogenases with high specific activity in Clostridium acetobutylicum. Inter J Hydrogen Energy 33(21): 6076-6081.
- Winkler, M., Kuhlgert, S., Hippler, M. and Happe, T. (2009). Characterization of the key step for light-driven hydrogen evolution in green algae. J Biol Chem 284(52): 36620-36627.
Article Information
Copyright
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
Category
Plant Science > Phycology > Protein
Biochemistry > Protein > Isolation and purification