A fusion tag to fold on: the S-layer protein SgsE confers improved folding kinetics to translationally fused enhanced green fluorescent protein.
Genetic fusion of two proteins frequently induces beneficial effects to the proteins, such as increased solubility, besides the combination of two protein functions. Here, we study the effects of the bacterial surface layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a on the folding of a C-terminally fused enhanced green fluorescent protein (EGFP) moiety. Although GFPs are generally unable to adopt a functional confirmation in the bacterial periplasm of Escherichia coli cells, we observed periplasmic fluorescence from a chimera of a 150-amino-acid N-terminal truncation of SgsE and EGFP. Based on this finding, unfolding and refolding kinetics of different S-layer-EGFP chimeras, a maltose binding protein-EGFP chimera, and sole EGFP were monitored using green fluorescence as indicator for the folded protein state. Calculated apparent rate constants for unfolding and refolding indicated different folding pathways for EGFP depending on the fusion partner used, and a clearly stabilizing effect was observed for the SgsE_C fusion moiety. Thermal stability, as determined by differential scanning calorimetry, and unfolding equilibria were found to be independent of the fused partner. We conclude that the stabilizing effect SgsE_C exerts on EGFP is due to a reduction of degrees of freedom for folding of EGFP in the fused state.
Department of NanoBiotechnology, Vienna Institute of BioTechnology, University fur Bodenkultur Wien, Vienna, Austria.
Robin
R
Ristl
Birgit
B
Kainz
Gerhard
G
Stadlmayr
Heinrich
H
Schuster
Dietmar
D
Pum
Paul
P
Messner
Christian
C
Obinger
Christina
C
Schaffer
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019897:Periplasm / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D058619:Maltose-Binding Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C055764:S-layer proteins; D049452:Green Fluorescent Proteins