Expression of the human UDP-glucuronosyltransferase UGT1*6 in Escherichia coli. Influence of bacterial signal peptides on the production and localization of the recombinant protein.
The membrane-bound human liver UDP-glucuronosyltransferase UGT1*6 was expressed in Escherichia coli. Exchange of the natural signal peptide by the bacterial signal peptides of pclB or OmpT proteins considerably increased the level of expression and, as the natural signal peptide, targeted the protein to the membranes. The extent of maturation of SpelB-UGT1*6 precursor was about 30%. No processing of sOmpT-UGT1*6 occurred but the processing rate of this precursor could be significantly increased by mutagenesis of the first two amino acid residues of the mature sequence. These expression vectors allowed us to produce high levels of recombinant mature UGT1*6 required for further structural studies.
Ouzzine
M
M
Centre du Médicament, URA CNRS no. 597, Faculté des Sciences Pharmaceutiques et Biologiques, Nancy, France.
Fournel-Gigleux
S
S
Pillot
T
T
Burchell
B
B
Siest
G
G
Magdalou
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects*; D005822:Genetic Vectors; D014453:Glucuronosyltransferase / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics* / Q000502:physiology; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis
D001426:Bacterial Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014453:Glucuronosyltransferase