Methods for the recombinant expression of active tyrosine kinase domains: Guidelines and pitfalls

Abstract

Protein tyrosine kinases (PTKs) are key signaling molecules and important drug targets. Although the efficient recombinant production of active PTKs is important for both pharmaceutical industry and academic research, most PTKs are still obtained from conventional, expensive and time-consuming insect-cell based expression. Host toxicity, kinase inactivity, insolubility and heterogeneity are among the reasons thought to preclude PTK expression in Escherichia coli. Herein we review these presumed roadblocks and their possible solutions for bacterial expression of PTKs, and give an overview on kinase activity assays. Finally, we report our experiences and observations with the kinases Src, Lyn and FAK as examples to illustrate implementation, effects and pitfalls of E. coli expression and in vitro assaying of PTKs.

Characterization of Phosphorylation Status and Kinase Activity of Src Family Kinases Expressed in Cell-Based and Cell-Free Protein Expression Systems

“…For tyrosine protein kinases in particular, problems such as low expression or the formation of inclusion bodies are frequently encountered. This might be due to the potential cytotoxicity of the tyrosine protein kinases or to misfolding [29,30]. Although large-scale expression of a human tyrosine kinase in E. coli is difficult, we have confirmed that all SFKs can be expressed at levels detectable by Western blotting (Figure 3A).…”

“…Non-specific phosphorylation of cellular proteins suggests that the functional heterologous kinase expression would alter the original functions of certain endogenous proteins. This may be one of the reasons for the difficulty of preparing human tyrosine kinases using E. coli hosts, as the toxicity of heterologous proteins in E. coli has been pointed out [30].…”

An Assay of Human Tyrosine Protein Kinase ABL Activity using an Escherichia Coli Protein Expression System

“…In particular, with tyrosine protein kinases, such problems as low expression or the formation of inclusion bodies are frequently encountered. This might be due to potential cytotoxicity of the tyrosine protein kinases or to misfolding [7,8]. To overcome these problems, insect or mammalian cells are frequently used in preparing recombinant tyrosine kinases.…”

Engineering soluble artificial epidermal growth factor receptor mimics capable of spontaneous in vitro dimerization

“…One of the factors that complicate functional studies of EGFR and the discovery of allosteric inhibitors is that full‐length EGFR family of receptors, that also contain HER2, HER3, and HER4 receptors, is challenging to obtain in a soluble form in vitro due to the difficulty of expressing transmembrane domains and the instability of the kinase domain (Diaz Galicia et al, 2019; Haacke et al, 2009). Truncated receptors are widely utilized to study the biochemical, structural, and functional aspects of this class of receptors (Aertgeerts et al, 2011; Jura et al, 2009; 2009; Kovacs, Das, et al, 2015; Kovacs, Zorn, et al, 2015; Littlefield et al, 2014; Zhang et al, 2006, 2007).…”

“…One of the factors that complicate functional studies of EGFR and the discovery of allosteric inhibitors is that full-length EGFR family of receptors, that also contain HER2, HER3, and HER4 receptors, is challenging to obtain in a soluble form in vitro due to the difficulty of expressing transmembrane domains and the instability of the kinase domain (Diaz Galicia et al, 2019;Haacke et al, 2009).…”