Bacterial lipid modification of proteins for novel protein engineering applications.
Functioning of proteins efficiently at the solid-liquid interface is critical to not only biological but also modern man-made systems such as ELISA, liposomes and biosensors. Anchoring hydrophilic proteins poses a major challenge in this regard. Lipid modification, N-acyl-S-diacylglyceryl-Cys, providing an 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 Escherichia 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, an 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 spectrometric 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.
Kamalakkannan
S
S
Centre for Biotechnology, Anna University, Chennai 600 025 and Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.
Murugan
V
V
Jagannadham
M V
MV
Nagaraj
R
R
Sankaran
K
K
D000595:Amino Acid Sequence; D001081:Apyrase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D008074:Lipoproteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D008566:Membranes / Q000378:metabolism; D015394:Molecular Structure; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012760:Shigella / Q000201:enzymology / Q000235:genetics
D017931:DNA Primers; D008074:Lipoproteins; D011994:Recombinant Proteins; D001081:Apyrase