Targeting the expression of functional murine CMP-sialic acid transporter to the E. coli inner membrane

The heterologous expression of functional mammalian integral membrane proteins still represents a significant hurdle towards their crystallization and structure elucidation. We have therefore explored the use of the OmpA signal sequence to deliberately target the expression of the murine CMP-sialic...

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Bibliographic Details
Published inBiochemical and biophysical research communications Vol. 362; no. 3; pp. 779 - 784
Main Authors Maggioni, Andrea, von Itzstein, Mark, Gerardy-Schahn, Rita, Tiralongo, Joe
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 26.10.2007
Subjects
60 APPLIED LIFE SCIENCES
Animals
Bacterial Outer Membrane Proteins - metabolism
Cell Membrane - metabolism
CELL MEMBRANES
Cloning, Molecular
CMP-sialic acid transporter
CRYSTALLIZATION
Cytidine Monophosphate N-Acetylneuraminic Acid - metabolism
E. coli
ESCHERICHIA COLI
Escherichia coli - metabolism
Genetic Techniques
Golgi Apparatus - metabolism
IN VITRO
LECITHINS
MEMBRANE PROTEINS
Mice
Models, Biological
N-Acetylneuraminic Acid - metabolism
OmpA signal sequence
Protein Structure, Tertiary
Recombinant Proteins - chemistry
SIALIC ACID
Sialic Acids - metabolism
SIGNALS
Spheroplasts - metabolism
Sialic acid
OmpA signal sequence
CMP-sialic acid transporter
E. coli
Membrane proteins
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Summary:The heterologous expression of functional mammalian integral membrane proteins still represents a significant hurdle towards their crystallization and structure elucidation. We have therefore explored the use of the OmpA signal sequence to deliberately target the expression of the murine CMP-sialic acid transporter, a Golgi-resident protein with 10 putative transmembrane domains, to the Escherichia coli inner membrane. Here, we show that the expression of an OmpA signal sequence-FLAG-CMP-sialic acid transporter fusion protein in E. coli results in the targeting and insertion of recombinant protein within the inner membrane. Significantly, functionality was confirmed by the ability of spheroplasted E. coli and mixed phosphatidylcholine– E. coli inner membrane proteoliposomes incorporating recombinant CMP-sialic acid transporter to accumulate CMP-sialic acid in vitro.
Bibliography:ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
ISSN:0006-291X
1090-2104
DOI:10.1016/j.bbrc.2007.08.070