Expression and purification of a novel rice (Oryza sativa L.) mitochondrial ATP synthase small subunit in Escherichia coli.
To clarify the function of the rice mitochondrial ATP synthase 6 kDa subunit (RMtATP6), a method of producing large quantities of this protein is needed. Here, we describe an Escherichia coli expression system for the rapid and economic expression of RMtATP6. The RMtATP6 gene (GenBank Accession No. ) was cloned into the pGEX-6p-3 vector to allow expression of RMtATP6 as a glutathione S-transferase (GST) fusion protein. The RMtATP6-GST fusion protein was purified by affinity chromatography using a glutathione-Sepharose 4B column. A Western blot analysis using anti-GST antibody showed that the fusion protein was not degraded. After enzymatic cleavage of the GST tail, the RMtATP6 protein showed a molecular weight of around 6 kDa. The predicted pI of this protein is 10.01. After improving the conditions of expression and the purification procedures, the final yield of the entire expression and purification process was about 4.6 mg of pure RMtATP6 protein per liter of bacterial culture.
Liu
Shenkui
S
Stress Molecular Biology Laboratory, Alkali Soil Natural Environmental Science Center (ASNESC), The Northeast Forestry University, Harbin, 150040, People's Republic of China. shenkuil@mail.nefu.edu.cn
Zhang
Xinxin
X
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics*; D015153:Blotting, Western; D002845:Chromatography; D004586:Electrophoresis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007544:Isopropyl Thiogalactoside / Q000737:chemistry; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry*; D012275:Oryza / Q000201:enzymology*; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry; D013696:Temperature
D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; D012965:Sodium Chloride; D005982:Glutathione Transferase; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases