Comparative expression study to increase the solubility of cold adapted Vibrio proteins in Escherichia coli.
Functional and structural studies require gene overexpression and purification of soluble proteins. We wanted to express proteins from the psychrophilic bacterium Vibrio salmonicida in Escherichia coli, but encountered solubility problems. To improve the solubility of the proteins, we compared the effects of six N-terminal fusion proteins (Gb1, Z, thioredoxin, GST, MBP and NusA) and an N-terminal His6-tag. The selected test set included five proteins from the fish pathogen V. salmonicida and two related products from the mesophilic human pathogen Vibrio cholerae. We tested the expression in two different expression strains and at three different temperatures (16, 23 and 37 degrees C). His6-tag was the least effective tag, and these vector constructs were also difficult to transform. MBP and NusA performed best, expressing soluble proteins with all fusion partners in at least one of the cell types. In some cases MBP, GST and thioredoxin fusions resulted in products of incorrect size. The effect of temperature is complex: in most cases level of expression increased with temperature, whereas the effect on solubility was opposite. We found no clear connection between the preferred expression temperature of the protein and the temperature of the original host organism's natural habitat.
Niiranen
Laila
L
The Norwegian Structural Biology Centre (NorStruct) and Department of Molecular Biotechnology, Institute of Medical Biology, Faculty of Medicine, University of Tromsø, N-9037 Tromsø, Norway.
Espelid
Sigrun
S
Karlsen
Christian R
CR
Mustonen
Milla
M
Paulsen
Steinar M
SM
Heikinheimo
Pirkko
P
Willassen
Nils P
NP
D044165:Aliivibrio salmonicida / Q000502:physiology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D003080:Cold Temperature; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D012995:Solubility; D040681:Structural Homology, Protein
D001426:Bacterial Proteins