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IJBT Vol.05 [2006] >
IJBT Vol.05(3) [July 2006] >
| Title: | Bacterial lipid modification in vitro: Synthetic peptide substrate for phosphatidylglycerol—Prolipoprotein diacylglyceryl transferase |
| Authors: | Selvan, A Tamil
Sankaran, K |
| Keywords: | bacterial lipid modification
signal sequence
peptide substrate
assay |
| Issue Date: | Jul-2006 |
| Publisher: | CSIR |
| IPC Code: | Int. Cl.8 C12N1/38 |
| Abstract: | Lipid modification is an emerging protein-engineering tool for providing hydrophobic anchor to hydrophilic proteins for immobilizing them onto a variety of man-made and biological surfaces. In this respect, N-acyl S-diacylglyceryl modification of N-terminal cysteine, called bacterial lipid modification has many advantages including the fact that it can be achieved through popular prokaryotic expression of proteins. The modification proceeds in a three-step cascade reaction in which three inner membrane enzymes, Phosphatidylglycerol:Prolipoprotein diacylglyceryl transferase, signal peptidase II and apolipoprotein N-acyl transferase, participate. Bacterial expression has limitations including the necessity of expensive downstream processing. However, in vitro modification has not been possible so far because the enzymology of this pathway is not well studied due to difficulty in assaying these enzymes. As a first step to overcome this problem we have designed the peptide substrate, MKATKSAVGSTLAGCSSHHHHHH, for in vitro lipid modification specifically the first enzyme, Phosphatidylglycerol:Prolipoprotein diacylglyceryl transferase, which catalyzes the committed step. The design was based on bioinformatics analysis of more than 1000 bacterial lipoprotein precursors. This synthetic peptide substrate was soluble and contained other built-in features useful in easier handling and purification. The designed substrate exhibited expected properties and in vitro diacylglyceryl modification was confirmed by Tricine SDS PAGE based mobility shift assay. |
| Page(s): | 327-331 |
| ISSN: | 0975-0967 (Online); 0972-5849 (Print) |
| Source: | IJBT Vol.05(3) [July 2006]
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