Different Fibrillation Behaviors of Lipase and Its Mutants

These fibrils have been the goal of many studies due to existence in neurodegenerative disease such as Parkinson s, Alzheimer s, and Huntington s and over expression of recombinant protein in bacteria host. The novel lipase that was isolated from a local Pseudomonas sp. was cloned and expressed in E.coli. This lipase forms fibrillar structure rapidly in physiological condition in the absence of urea or any denaturants. This feature of lipase makes it a good candidate for fibrils studies. Glutamic acid residues at position 171 and 28 of N-terminus of this protein were opted for investigation of fibril formation. The mutants with single point mutation in glutamic acid residue and deletion in the first 28 residue were cloned and express in E.coli. These proteins were purified using nickel agarose column and their fibrillation characterization were compared using Congo red assay and thioflavin T fluorescent assay with the native one. This comparison shows although none of the proteins have activity after purification in the absence of denaturants but the behavior of their fibrillation is really different. The mutant with single point mutation forms fibrillar structure with urea dilution but the amounts of it decreased considerably. In this work the mechanism of Lipase fibrillation is suggested.

کلیدواژه ها:

Fibrillation ، Lipase ، ThT assay ، Recombinant Protein Production