Insertion of carrier proteins into hydrophilic loops of the Escherichia coli lactose permease.
We describe the design and characterization of a set of fusion proteins of the Escherichia coli lactose (lac) permease in which a set of five different soluble "carrier" proteins (cytochrome(b562), flavodoxin, T4 lysozyme, beta-lactamase and 70 kDa heat shock ATPase domain) were systematically inserted into selected loop positions of the transporter. The design goal was to increase the exposed hydrophilic surface area of the permease, while minimizing the internal flexibility of the resulting fusion proteins in order to improve the crystallization properties of the membrane protein. Fusion proteins with insertions into the central hydrophilic loop of the lac permease were active in transport lactose, although only the fusion proteins with E. coli cytochrome(b562), E. coli flavodoxin or T4 lysozyme were expressed at near wild-type lac permease levels. Eight other loop positions were tested with these three carriers, leading to the identification of additional fusion proteins that were active and well-expressed. By combining the results from the single carrier insertions, we have expressed functional "double fusion" proteins containing cytochrome(b562) domains inserted in two different loop positions.
Engel
Christian K
CK
Division of Molecular and Structural Biology, Ontario Cancer Institute, Toronto, Canada.
Chen
Lu
L
Privé
Gilbert G
GG
D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D027981:Symporters
D002352:Carrier Proteins; D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D027981:Symporters; C017850:cytochrome b562, E coli; C021499:lactose permease