Abstract
This protocol describes the heterologous expression and purification of proteins related to anoxic hydrogen production of Chlamydomonas reinhardtii (Noth et al., 2013). For this, the bacterial expression hosts Escherichia coli BL21 (DE3) ΔiscR (Akhtar MK et al., 2008) and Clostridium acetobutylicum ATCC 824 are used, which are grown either aerobic or anaerobic with glucose. Two standard chromatographic methods for purification were applied using His- and StrepII-tagged proteins (Figure 1). All procedures have been performed in an anaerobic tent to avoid the access of oxygen.
Keywords: Fermentation, Protein Isolation, Hydrogenase, Iron Sulfur Cluster, Chlamydomonas reinhardtii
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
Equipment
Procedure
Recipes
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
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