Comparative analysis of protein expression systems and PTM landscape in the study of transcription factor ELK-1

• Published in: Protein expression and purification Vol. 203; p. 106216
• Main Authors: Ducker, Charles, Ratnam, Manohar, Shaw, Peter E., Layfield, Robert
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2023
• Subjects: Acetylation; Animals; DNA - metabolism; ELK-1; Escherichia coli - metabolism; ets-Domain Protein Elk-1 - biosynthesis; ets-Domain Protein Elk-1 - metabolism; HEK293 Cells; Humans; Mammals; Mitogens; Phosphorylation; Protein expression; Protein Processing, Post-Translational; Receptors, Androgen - metabolism; Sf9 Cells - metabolism; Transcription factors; Transcription Factors - metabolism; ELK-1; Protein expression; Phosphorylation; Transcription factors; Acetylation; Mitogens
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2022.106216

Post-translational modifications (PTMs) are important for protein folding and activity, and the ability to recreate physiologically relevant PTM profiles on recombinantly-expressed proteins is vital for meaningful functional analysis. The ETS transcription factor ELK-1 serves as a paradigm for cellular responses to mitogens and can synergise with androgen receptor to promote prostate cancer progression, although in vitro protein function analyses to date have largely overlooked its complex PTM landscapes. We expressed and purified human ELK-1 using mammalian (HEK293T), insect (Sf9) and bacterial (E. coli) systems in parallel and compared PTMs imparted upon purified proteins, along with their performance in DNA and protein interaction assays. Phosphorylation of ELK-1 within its transactivation domain, known to promote DNA binding, was most apparent in protein isolated from human cells and accordingly conferred the strongest DNA binding in vitro, while protein expressed in insect cells bound most efficiently to the androgen receptor. We observed lysine acetylation, a hitherto unreported PTM of ELK-1, which appeared highest in insect cell-derived ELK-1 but was also present in HEK293T-derived ELK-1. Acetylation of ELK-1 was enhanced in HEK293T cells following starvation and mitogen stimulation, and modified lysines showed overlap with previously identified regulatory SUMOylation and ubiquitination sites. Our data demonstrate that the choice of recombinant expression system can be tailored to suit biochemical application rather than to maximise soluble protein production and suggest the potential for crosstalk and antagonism between different PTMs of ELK-1. •Recombinant human ELK-1 protein was purified following expression in bacterial, insect and mammalian cells.•ELK-1 showed highest phosphorylation levels and DNA-binding when expressed in human (HEK293T) cells.•ELK-1 produced in insect (Sf9) cells showed strongest interaction to the disease-relevant cofactor Androgen Receptor.•Newly identified ELK-1 lysine acetylation sites overlap with established SUMOylation and ubiquitination targets.