Uncovering the role of leucine 59 in Renilla luciferase stability and activity with error-prone PCR: Implications for protein engineering

Zahra Fanaei-Kahrani; Rahman Emamzadeh; Mahboobeh Nazari

doi:10.1016/j.pep.2023.106378

Uncovering the role of leucine 59 in Renilla luciferase stability and activity with error-prone PCR: Implications for protein engineering

Protein Expr Purif. 2024 Feb:214:106378. doi: 10.1016/j.pep.2023.106378. Epub 2023 Oct 8.

Authors

Zahra Fanaei-Kahrani¹, Rahman Emamzadeh², Mahboobeh Nazari³

Affiliations

¹ Department of Cell and Molecular Biology and Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran.
² Department of Cell and Molecular Biology and Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran. Electronic address: r.emamzadeh@sci.ui.ac.ir.
³ Endocrine Research Center, Institute of Endocrinology and Metabolism, Iran University of Medical Sciences, Tehran, Iran; Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.

PMID: 37816476
DOI: 10.1016/j.pep.2023.106378

Abstract

A new variant of Renilla luciferase, named Met C-SRLuc 8, was obtained from a random mutagenesis library and expressed in Escherichia coli BL21 (DE3) plys and purified. The results of the enzyme's binding affinity, kinetic stability, and bioinformatic studies demonstrated that leucine 59, located within the hot-spot foldon in the N-terminal domain of the protein, plays a significant role in the stability and activity of Renilla luciferase. These findings may facilitate the engineering of different variants of this enzyme to achieve thermally stable versions for various biotechnological applications.

Keywords: Enzyme kinetics; Leucine 59; Protein engineering; Renilla luciferase.

MeSH terms

Biotechnology*
Leucine
Luciferases, Renilla / chemistry
Polymerase Chain Reaction
Protein Engineering*

Substances

Luciferases, Renilla
Leucine