A new strategy to produce active human Src from bacteria for biochemical study of its regulation

doi:10.1016/j.bbrc.2006.05.180

Biochemical and Biophysical Research Communications

Volume 346, Issue 2, 28 July 2006, Pages 606-611

https://doi.org/10.1016/j.bbrc.2006.05.180 Get rights and content

Abstract

Enzymological studies of Src protein tyrosine kinase have been hindered by the lack of a suitable bacterial expression system. Poor expression of active Src appears to be due to toxicity associated with its kinase activity. To overcome this problem, we fused Src to a protein tyrosine phosphatase with an affinity tag and an appropriate thrombin cleavage site. Upon affinity purification of the fusion protein, Src was released by thrombin digestion and further purified by FPLC. This strategy has been used to produce several Src mutants that display catalytic and regulatory properties similar to those from eukaryotic expression systems. Characterization of the Src mutants confirmed that inactivation of Src by Csk through tail tyrosine phosphorylation required the Src SH3 domain.

Section snippets

Materials and methods

Construction of human Src expression plasmids. Construction of wild type human Src expression plasmid was achieved in three steps. First, the human PTP1B cDNA encoding the region of Met1 to Gly322 was amplified by polymerase chain reaction. The cDNA fragment, containing a BamHI site at 5′-end and consecutive XbaI and SalI sites at 3′-end, was cloned into pMAL c2x expression vector (New England Biolabs) through BamHI and SalI cloning sites. Second, an adaptor encoding a thrombin cleavage site

Results

To test the hypothesis that the difficulty of expressing Src in bacteria was due to Src phosphorylating bacterial proteins, thus resulting in bacterial toxicity, we determined if introducing a protein tyrosine phosphatase (PTP) activity would overcome this apparent kinase activity-triggered problem. It was hoped that the PTP would dephosphorylate any bacterial protein that might be phosphorylated by Src and prevent any toxicity caused by Src kinase activity. There were several options to

Discussion

Many factors contribute to the difficulty of expressing a foreign protein in bacteria. Such factors include incorrect folding leading to aggregation, uncommon codon usage leading to low yield of expression, and protein degradation leading to low yield and impurity in purification. The inability to express active Src did not seem to be caused by any of these common reasons, since kdSrc can be highly expressed and well purified [10], and retains the molecular dynamics necessary for Src regulation

Acknowledgments

This work was supported by Grants from the American Cancer Society (RSG-04-247-01-CDD) and NIH (1 RO1 CA111687, and 1 P20 RR16457). FPLC was provided by RI-INBRE Centralized Research Core Facility. DNA sequencing was done at URI Genomics and Sequencing Center.

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Cited by (19)

Methods for the recombinant expression of active tyrosine kinase domains: Guidelines and pitfalls
2019, Methods in Enzymology
Citation Excerpt :
PTP1B had earlier been identified as the primary human phosphatase targeting and activating c-Src (Bjorge, Pang, & Fujita, 2000) and other PTKs (Fan, Lin, Lucito, & Tonks, 2013) and was used to improve yield and homogeneity of recombinant c-Src, c-Abl and c-Met KDs (Wang, Marimuthu, et al., 2006). Phosphatase co-expression was then reported in subsequent studies using either PTP1B (Cui & Sun, 2018; Tu, Wang, Cai, Zhou, & Zhang, 2014; Wang, Ayrapetov, Lin, & Sun, 2006) or YopH (Albanese et al., 2018; Filippakopoulos et al., 2008; Wilson et al., 2015). With one dissenting opinion (Wang, Marimuthu, et al., 2006), there seems to be a consensus that the active Src KD is cytotoxic to E. coli and that its recombinant expression benefits from phosphatase co-expression.
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.
The N2-Src neuronal splice variant of C-Src has altered SH3 domain ligand specificity and a higher constitutive activity than N1-Src
2015, FEBS Letters
Citation Excerpt :
The expression of active Src kinases in E. coli is problematic, due to the toxicity of tyrosine phosphorylation in bacteria that do not support this form of post-translational modification [35]. To overcome this issue we fused a cleavable tyrosine phosphatase (PTP1B) catalytic domain to the N-terminus of the kinase as previously reported (Fig. 1A; [36]). This modification yielded sufficient pure active kinase to perform in vitro kinase assays (Fig. 1B).
N2-Src is a poorly understood neuronal splice variant of the ubiquitous C-Src tyrosine kinase, containing a 17 amino acid insert in its Src homology 3 (SH3) domain. To characterise the properties of N2-Src we directly compared its SH3 domain specificity and kinase activity with C- and N1-Src in vitro. N2- and N1-Src had a similar low affinity for the phosphorylation of substrates containing canonical C-Src SH3 ligands and synaptophysin, an established neuronal substrate for C-Src. N2-Src also had a higher basal kinase activity than N1- and C-Src in vitro and in cells, which could be explained by weakened intramolecular interactions. Therefore, N2-Src is a highly active kinase that is likely to phosphorylate alternative substrates to C-Src in the brain.
Bacterial expression and purification of active hematopoietic cell kinase
2011, Protein Expression and Purification
Citation Excerpt :
However, the yield of purified soluble Hck was still very small (∼0.02 mg/L cell culture), but increased by more than 10-fold (to ∼0.25 mg/L cell culture) when Hck was co-expressed with YopH phosphatase (Table 2, Fig. 3). Similar yields have been reported for a construct of Src, comprising the SH3, SH2 and kinase domains and fused to PTP1B phosphatase [23], but are by a large margin lower than yields obtained for a kinase dead mutant of this construct or for the isolated Src kinase domain. These quantities might represent the upper limit that can be obtained for an active multi-domain SFK construct from E. coli.
Src family kinases (SFKs) are traditionally purified from eukaryotic expression systems. These expression systems can be costly, yield heterogeneously phosphorylated protein samples and present difficulties when metabolic labeling is required for structural studies. Therefore, many attempts have been made to develop bacterial purification systems for SFKs. So far, high-yield bacterial expression systems have only been achieved for SFK kinase domains or for inactive mutants of constructs containing the regulatory SH3 and SH2 domains, but not for their active forms. Herein described is a bacterial expression system for the wild type, active SFK Hck containing SH3, SH2 and kinase domains. Hck plays an important role in phagocyte function as well as the etiology of chronic myeloid leukemia as Hck is an interaction partner of Bcr-Abl. Structural studies of Hck are essential to fully understand the signaling processes involved in host defense and leukemogenesis. Successful bacterial expression of Hck was possible by a dual strategy: (1) co-expression with YopH phosphatase in order to control host toxicity, and (2) expression in a bacterial strain that is RNase E deficient, which dramatically increased overall expression levels. The expressed Hck construct is unphosphorylated and appears to be in an open conformation. Bacterially expressed Hck is capable of autophosphorylation, phosphorylates substrate at rates comparable to insect cell expressed Hck, and can be inhibited by staurosporine and Csk.
Identification of N-Terminal Lobe Motifs that Determine the Kinase Activity of the Catalytic Domains and Regulatory Strategies of Src and Csk Protein Tyrosine Kinases
2009, Journal of Molecular Biology
Csk and Src protein tyrosine kinases are structurally homologous but use opposite regulatory strategies. The isolated catalytic domain of Csk is intrinsically inactive and is activated by interactions with the regulatory Src homology 3 (SH3) and SH2 domains, while the isolated catalytic domain of Src is intrinsically active and is suppressed by interactions with the regulatory SH3 and SH2 domains. The structural basis for why one isolated catalytic domain is intrinsically active while the other is inactive is not clear. In this study, we identified structural elements in the N-terminal lobe of the catalytic domain that render the Src catalytic domain active. These structural elements include the α-helix C region, a β turn between the β4 and β5 strands, and an Arg residue at the beginning of the catalytic domain. These three motifs interact with one another to activate the Src catalytic domain, but the equivalent motifs in Csk directly interact with the regulatory domains that are important for Csk activation. The Src motifs can be grafted to the Csk catalytic domain to obtain an active Csk catalytic domain. These results, together with available Src and Csk tertiary structures, reveal an important structural switch that determines the kinase activity of a catalytic domain and dictates the regulatory strategy of a kinase.
Bacterial expression and purification of Interleukin-2 Tyrosine kinase: Single step separation of the chaperonin impurity
2008, Protein Expression and Purification
Biochemical and biophysical characterization of kinases requires large quantities of purified protein. Here, we report the bacterial expression and purification of active Itk kinase domain (a Tec family kinase) using ArcticExpress cells that co-express the chaperonin system Cpn60/10 from Oleispira antarctica. We describe a simple one step MgCl₂/ATP/KCl incubation procedure to remove the co-purifying chaperonin impurity. Chaperonin co-purification is a common problem encountered during protein purification and the simple incubation step described here completely overcomes this problem. The approach targets the chaperonin system rather than the protein of interest and is therefore widely applicable to other protein targets.
N-terminal cysteinyl proteins can be prepared using thrombin cleavage
2008, FEBS Letters
Expressed protein ligation – which allows native proteins to be selectively linked together by a normal peptide bond in an aqueous environment – has emerged as a powerful technique. The technique requires the formation of a C-terminal α-thioester and an N-terminal Cys. An N-terminal Cys can be formed by enzymatic cleavage, commonly using the Factor Xa and TEV proteases. We show that thrombin can be used for the formation of N-terminal Cys, providing another choice of reagents for expressed protein ligation. Proteins with N-terminal Cys can be obtained by the convenient modification of vectors with the putative thrombin cleavage site LVPRG to LVPRC. Two example protein domains (Csk and Abl tyrosine kinase domain) with N-terminal Cys are demonstrated using this method. The use of thrombin protease to generate N-terminal Cys overcomes some of the limitations of existing methods, making it generally useful for expressed protein ligation and other biotechnological applications.

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^☆: Abbreviations: PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; MBP, maltose binding protein; Src-ΔSH3, a Src mutant lacking the SH3 domain; Src-NT, a Src mutant lacking the C-terminal tail; Csk, C-terminal Src kinase.

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A new strategy to produce active human Src from bacteria for biochemical study of its regulation☆

Abstract

Section snippets

Materials and methods

Results

Discussion

Acknowledgments

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

Methods Enzymol.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

Arch. Biochem. Biophys.

J. Mol. Biol.

Regulation, substrates and functions of Src

Biochem. Biophys. Acta

Src tyrosine kinase is a novel direct effector of G proteins

Cell