The present invention is to make use of bacterial lipid modification of proteins to solve the anchorage problem, as successfully addressed by bacteria. The immobilization of bacterial lipid modified Hu-IFN-? protein (highly hydrophilic protein) to hydrophobic surface of ELISA plates. The mature Hu-IFN-? containing His-tag was cloned in an in-house developed vector pLMAII, after the signal sequence of Braun's lipoprotein. The engineered Hu-IFN-? construct was expressed under T7 expression system in an arabinose-inducible E. coli BL21 AL The membrane localization of the expressed lipoprotein together with the slower mobility compared to native forms on a Tricine-SDS-PAGE were first-level indications for lipid-modification. Further, [9, 10- 3H] palmitic acid-labeling of Hu-IFN-? variants and E. coli BL21AI, that served as host control revealed much of radioactivity associated only with lipid-modified Hu-IFN-?, confirming bacterial lipid modification of Hu-IFN-?.
The below formula shows the lipid structure, which imparts the hydrophobic character to the engineered protein where the diacylglyceryl group attached through a thioether linkage to the Sulphydryl of the N-terminal Cys and an acyl group attached through amide linkage to the amino terminal of Cys. The three acyl moieties impart hydrophobicity and contribute to highly efficient binding of the engineered protein, in this case, lipid-modified Hu-IFN- ?.
