Solubility of recombinant Src homology 2 domains expressed in E. colican be predicted by TANGO

Thorny Cecilie Bie Andersen,Per Eugen Kristiansen,Kjersti Lindsjø,Anne Spurkland,Amy H Andreotti,Lars Skjeldal,Cecilie Dahl Hem,Lise Koll

doi:10.1186/1472-6750-14-3

Thorny Cecilie Bie Andersen, Per Eugen Kristiansen + Show 6 more

Open Access

https://doi.org/10.1186/1472-6750-14-3

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Abstract

BackgroundSignalling proteins often contain several well defined and conserved protein domains. Structural analyses of such domains by nuclear magnetic spectroscopy or X-ray crystallography may greatly inform the function of proteins. A limiting step is often the production of sufficient amounts of the recombinant protein. However, there is no particular way to predict whether a protein will be soluble when expressed in E.coli. Here we report our experience with expression of a Src homology 2 (SH2) domain.ResultsThe SH2 domain of the SH2D2A protein (or T cell specific adapter protein, TSAd) forms insoluble aggregates when expressed as various GST-fusion proteins in Escherichia coli (E. coli). Alteration of the flanking sequences, or growth temperature influenced expression and solubility of TSAd-SH2, however overall yield of soluble protein remained low. The algorithm TANGO, which predicts amyloid fibril formation in eukaryotic cells, identified a hydrophobic sequence within the TSAd-SH2 domain with high propensity for beta-aggregation. Mutation to the corresponding amino acids of the related HSH2- (or ALX) SH2 domain increased the yield of soluble TSAd-SH2 domains. High beta-aggregation values predicted by TANGO correlated with low solubility of recombinant SH2 domains as reported in the literature.ConclusionsSolubility of recombinant proteins expressed in E.coli can be predicted by TANGO, an algorithm developed to determine the aggregation propensity of peptides. Targeted mutations representing corresponding amino acids in similar protein domains may increase solubility of recombinant proteins.

Highlights

Signalling proteins often contain several well defined and conserved protein domains
Small amounts of recombinant Glutathion S-transferase (GST)-T cell specific adapter protein (TSAd) Src homology 2 (SH2) was present in the lysate, most of this soluble fraction could be captured on glutathione sepharose beads
Anti-GST-immunoblotting of equal amounts of the soluble fractions compared to a defined amount of purified, soluble GST (Figure 2C,i), revealed that the yield of the TSAd SH2 domain) was only 0,15 mg/l bacterial cell culture, while that of Lck-SH2 was estimated to be close to 20 mg/l bacterial culture (Figure 2D)

Summary

Introduction

Signalling proteins often contain several well defined and conserved protein domains. Structural analyses of such domains by nuclear magnetic spectroscopy or X-ray crystallography may greatly inform the function of proteins. We report our experience with expression of a Src homology 2 (SH2) domain. Src homology 2 (SH2) are structurally conserved protein domains of approximately100 amino acids (aa). SH2 domains regulate numerous intracellular signal transduction events through interaction with tyrosine phosphorylated proteins [1]. The five strands of the central beta sheet separate a conserved phosphotyrosine binding pocket from a more variable pocket that typically binds the third amino acid C-terminal to the phosphorylated tyrosine. Structural analysis of the SH2D1A gene product, Slam associated protein (SAP), revealed the structural basis for a phosphotyrosine independent three-pronged gene SH2D2A SH2D2A SH2D2A HSH2 Lck species aa homo 95 mouse 116 rat 116 homo 33 homo 126

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