Application of Mistic to improving the expression and membrane integration of histidine kinase receptors from Escherichia coli.
Integral membrane proteins have become the focus of interest of many laboratories and structural genomics consortia, but their study is hampered by bottlenecks in production, solubilization, purification and crystallization. In our laboratory we have addressed the problem of high-level protein expression in the membrane of Escherichia coli by use of Mistic, a novel Bacillus subtilis protein, as a fusion partner. In this study we examine the effect of Mistic on protein expression and membrane integration levels of members of the E. coli histidine kinase receptor family. We find that Mistic fusion invariably increases the overall yield by targeting the cargo proteins more efficiently to the membrane and may even replace the signal sequence. Mistic fusion methods will likely be instrumental for high-level expression of other integral membrane proteins.
Kefala
Georgia
G
Structural Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Rd, La Jolla, CA 92037, USA.
Kwiatkowski
Witek
W
Esquivies
Luis
L
Maslennikov
Innokentiy
I
Choe
Senyon
S
D001412:Bacillus subtilis / Q000737:chemistry*; D015153:Blotting, Western; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D000071677:Histidine Kinase; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases; D000071677:Histidine Kinase