Effect of C-terminal His-tag and Purification Routine on the Activity and Structure of the Metalloenzyme, L-alanyl-d-glutamate Peptidase of the Bacteriophage T5
Document Type
Article
Publication Date
2019
Keywords
l-Alanyl-d-glutamate peptidase, Zinc binding, His-tag, Structural properties, Conformational stability, Enzymatic activity
Digital Object Identifier (DOI)
https://doi.org/10.1016/j.ijbiomac.2018.11.219
Abstract
In this work, we studied the effect of the C-terminally attached poly-histidine tag (His-tag), as well as the peculiarities of the protein purification procedure by the immobilized metal affinity chromatography (IMAC) on the activity and structure of the metalloenzyme, l-alanyl-d-glutamate peptidase of bacteriophage T5 (EndoT5), whose zinc binding site and catalytic aspartate are located near the C-terminus. By itself, His-tag did not have a significant effect on either activity or folding of the polypeptide chain, nor on the binding of zinc and calcium ions to the protein. However, the His-tagged EndoT5 samples had low shelf-life, with storage of these samples resulting in an increased propensity for protein self-association and decreased enzymatic activity of EndoT5. Furthermore, disastrous effects on the activity of the enzyme were exerted by the presence of imidazole and nickel ions accompanying metal chelate chromatography. The activity of the protein can be restored by thorough washing off of these low molecular impurities via the prolonged dialysis of the His-tagged EndoT5 samples at the specifically elaborated conditions.
Was this content written or created while at USF?
Yes
Citation / Publisher Attribution
International Journal of Biological Macromolecules, v. 124, p. 810-818
Scholar Commons Citation
Kutyshenko, Victor P.; Mikoulinskaia, Galina V.; Chernyshov, Sergei V.; Yegorov, Alexander Y.; Prokhorov, Dmitry A.; and Uversky, Vladimir N., "Effect of C-terminal His-tag and Purification Routine on the Activity and Structure of the Metalloenzyme, L-alanyl-d-glutamate Peptidase of the Bacteriophage T5" (2019). Molecular Medicine Faculty Publications. 131.
https://digitalcommons.usf.edu/mme_facpub/131
