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Biochemical and Biophysical Research Communications, Vol.362, No.3, 779-784, 2007
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 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. (c) 2007 Elsevier Inc. All rights reserved.