A modified method for the purification of active large enzymes using the glutathione S-transferase expression system.
The glutathione S-transferase (GST) fusion protein system is widely used for high-level expression and efficient purification of recombinant proteins from bacteria. However many GST-tagged proteins are insoluble, and the existing procedures, which employ a mixture of detergents to solubilize the molecules, frequently compromise their functional activity. A further limitation is that large proteins (>80 kDa) are poorly isolated by the current methods and are contaminated by truncated forms. To overcome these problems, we provide here an improved method for efficient purification of active large GST-tagged enzymes such as the 180-kDa GST-fused mitochondrial RNA polymerase.
Dipartimento di Biochimica e Biologia Molecolare "Ernesto Quagliariello," Università degli Studi di Bari Aldo Moro, 70125 Bari, Italy.
Stefania
S
Deceglie
Claudia
C
Lionetti
Marina
M
Roberti
Palmiro
P
Cantatore
Paola
P
Loguercio Polosa
D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D012321:DNA-Directed RNA Polymerases