Heterologous Production and Anaerobic Purification of His- and StrepII-tagged Recombinant Proteins
融合His及StrepII标签的莱茵衣藻蛋白的原核表达及纯化
发布: 2013年09月05日第3卷第17期 DOI: 10.21769/BioProtoc.881 浏览次数: 15573
评审: Ru Zhang
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参见作者原研究论文
The authors used this protocol in:
Feb 2013
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 (发酵)
Protein Isolation (蛋白质分离)
Hydrogenase (氢化酶)
Iron Sulfur Cluster (铁硫簇)
Chlamydomonas reinhardtii (莱茵衣藻)
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
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Noth, J. (2013). Heterologous Production and Anaerobic Purification of His- and StrepII-tagged Recombinant Proteins . Bio-protocol 3(17): e881. DOI: 10.21769/BioProtoc.881.
- 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.
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分类
植物科学 > 藻类学 > 蛋白质
生物化学 > 蛋白质 > 分离和纯化
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