Importance of the Choice of a Recombinant System to Produce Large Amounts of Functional Membrane Protein hERG.
Fiche publication
Date publication
juin 2019
Journal
International journal of molecular sciences
Auteurs
Membres identifiés du Cancéropôle Est :
Dr WAGNER Renaud
Tous les auteurs :
Vasseur L, Cens T, Wagner R, Saint N, Kugler V, Chavanieu A, Ouvry C, Dupré C, Ferry G, Boutin JA
Lien Pubmed
Résumé
Human ether-a-gogo related gene (hERG) product is the membrane potassium channel Kv11.1, which is involved in the electrical activity of the heart. As such, it is a key player in the toxicity of many drug candidates. Therefore, having this protein at hand during earlier stages of drug discovery is important for preventing later toxicity. Furthermore, having a fair quantity of functional channels may help in the development of the necessary techniques for gaining insight in this channel structure. Thus, we performed a comparative study of methods for over-expressing a mutated but functional, hERG in different orthologous hosts, such as yeast, bacteria, insect and human cell lines. We also engineered the protein to test various constructs of a functional channel. We obtained a significant amount of a functional mutant channel from HEK cells that we thoroughly characterized. The present work paves the way for the expression of large amounts of this protein, with which protein crystallization or cryo-electronic microscopy will be attempted. This will be a way to gain information on the structure of the hERG active site and its modelization to obtain data on the pauses of various reference compounds from the pharmacopeia, as well as to gain information about the thermodynamics of the hERG/ligand relationship.
Mots clés
E. coli, HEK cells, Kv11.1, P. pastoris, baculovirus expression system, expression, functional characterization, hERG, purification
Référence
Int J Mol Sci. 2019 Jun 28;20(13):