An efficient method for the preparation of preferentially heterodimerized recombinant S100A8/A9 coexpressed in Escherichia coli

Junichiro Futami,Yuki Atago,Akari Azuma,Endy Widya Putranto,Rie Kinoshita,Hitoshi Murata,Masakiyo Sakaguchi

doi:10.1016/j.bbrep.2016.03.009

Junichiro Futami, Yuki Atago + Show 5 more

Open Access

https://doi.org/10.1016/j.bbrep.2016.03.009

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Journal: Biochemistry and Biophysics Reports	Publication Date: Mar 19, 2016
Citations: 18	License type: cc-by

Affiliation: Okayama University

Abstract

It is now known that multicomponent protein assemblies strictly regulate many protein functions. The S100 protein family is known to play various physiological roles, which are associated with alternative complex formations. To prepare sufficient amounts of heterodimeric S100A8 and S100A9 proteins, we developed a method for bicistronic coexpression from a single-vector system using Escherichia coli cells as a host. The complex formation between S100A8 and S100A9 appears to be dependent on the thermodynamic stability of the protein during expression. The stable S100A8/A9 heterodimer complex spontaneously formed during coexpression, and biologically active samples were purified by cation-exchange chromatography. Semi-stable homodimers of S100A8 and S100A9 were also formed when expressed individually. These results suggest that the assembly of S100 protein complexes might be regulated by expression levels of partner proteins in vivo. Because protein assembly occurs rapidly after protein synthesis, coexpression of relevant proteins is crucial for the design of multicomponent recombinant protein expression systems.

Highlights

It is known that multicomponent protein assemblies strictly regulate many protein functions
The gene fragments encoding human S100A8 (Uniprot: P05109) and S100A9 (Uniprot: P06702) were prepared by GeneArt Strings DNA fragment gene synthesis service (Life Technologies) with the sequence optimized for E. coli protein expression
The solubilization solution containing DTT at pH 7.5 was crucial for resuspending precipitated S100A8 and S100A9 proteins, because both proteins possess one reactive Cys, making it easy to form disulfide-bound homodimers, which are frequently observed for S100A9 under non-reducing conditions

Summary

Introduction

Heterodimerization from semi-stable homodimers is not a spontaneous process because it requires external energy to dissociate the homodimers into monomers. Because of this property, cofolding or coexpression of heterodimerizing S100 proteins is a reasonable strategy to yield stable heterodimer. J. Futami et al / Biochemistry and Biophysics Reports 6 (2016) 94–100 a coexpression strategy [13], utilizing a bicistronic plasmid DNA expression system. Futami et al / Biochemistry and Biophysics Reports 6 (2016) 94–100 a coexpression strategy [13], utilizing a bicistronic plasmid DNA expression system This simplified methodology allows one to obtain large quantities of biologically active heterodimeric S100A8/A9 protein from E. coli, and purified it using single step ion-exchange chromatography

Materials and methods

Protein expression and purification

Analysis of molecular complex by size-exclusion HPLC

Result

Findings

Discussion