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PEDS Advance Access published online on November 19, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh087
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Received April 22, 2004
Revised September 22, 2004
Accepted October 21, 2004

Article

Bacterial lipid modification of proteins for novel protein engineering applications

S. Kamalakkannan 1, V. Murugan 1, M. V. Jagannadham 2, R. Nagaraj 2, and K. Sankaran 1*

1 Centre for Biotechnology, Anna University, Chennai - 600 025, India
2 Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad - 500 007, India

* To whom correspondence should be addressed.
K. Sankaran, E-mail: ksankran{at}yahoo.com


  Abstract

Functioning of proteins efficiently at the solid-liquid interface is critical to not only biological but also modern man-made systems like ELISA, liposomes and biosensors. Anchoring hydrophilic proteins poses a major challenge in this regard. Lipid modification, N-acyl-S-diacylglyceryl-Cys, providing N-terminal hydrophobic membrane anchor is a viable solution that bacteria have successfully evolved but remains unexploited. Based on the current understanding of this ubiquitous and unique bacterial lipid modification it is possible to use E.coli, the popular recombinant protein expression host, for converting a non-lipoprotein to a lipoprotein with a hydrophobic anchor at the N-terminal end. We report two strategies applicable to non-lipoproteins (with or without signal sequences) employing minimal sequence change. Taking periplasmic Shigella Apyrase as an example its signal sequence was engineered to include a lipobox, essential determinant for lipid modification, or its mature sequence was fused to the signal sequence of abundant outer membrane lipoprotein, Lpp. Lipid modification was proved by membrane localization, electrophoretic mobility shift and mass-spec analysis. Substrate specificity and specific activity measurements indicated functional integrity after modification. In conclusion, a convenient protein-engineering strategy for converting non-lipoprotein to lipoprotein for commercial application has been devised and tested successfully.

Keywords: Protein Engineering; Bacterial Lipid Modification; Apyrase; Signal Sequence.
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