Effect of N-terminal solubility enhancing fusion proteins on yield of purified target protein
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We have studied the effect of solubilising N-terminal fusion proteins on the yield of target protein after removal of the fusion partner and subsequent purification using immobilised metal ion affinity chromatography. We compared the yield of 45 human proteins produced from four different expression vectors: three having an N-terminal solubilising fusion protein (the GB1-domain, thioredoxin, or glutathione S-transferase) followed by a protease cleavage site and a His tag, and one vector having only an N-terminal His tag. We have previously observed a positive effect on solubility for proteins produced as fusion proteins compared to proteins produced with only a His tag in Escherichia coli. We find this effect to be less pronounced when we compare the yields of purified target protein after removal of the solubilising fusion although large target-dependent variations are seen. On average, the GB1+His fusion gives significantly higher final yields of protein than the thioredoxin+His fusion or the His tag, whereas GST+His gives lower yields. We also note a strong correlation between solubility and target protein size, and a correlation between solubility and the presence of peptide fragments that are predicted to be natively disordered.
KeywordsEscherichia coli fusion protein high throughput His tag solubility structural genomics
enhanced green fluorescent protein
immobilised metal ion affinity chromatography
maltose binding protein
2-(N-morpholino) ethanesulfonic acid
open reading frame
ribosome binding site
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We would like to acknowledge the contributions of John Löfblom and Kristina Bergström for initial cloning of genes 202 to 215, Harry Brumer and Hongbin Henriksson for the mass spectrometry analysis and Susanne van den Berg for construction of the vectors pTH8 and pTH18. This work was supported by the European Commission Integrated Project SPINE (QLG2-CT-2002-00988) as part of the Framework 5 Quality of Life and Management of Living Resources Program, by the Wallenberg Consortium North (WCN) and by the Swedish foundation for strategic research (SSF).
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