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

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.

Keywords: Calprotectin; Coexpression; Heterodimerization; S100 protein.

Graphical abstract

Fig. 1

Schema for construction of S100A8/A9 coexpression vector and their protein expression in E. coli. (A) Expression vector for S100A8 or S100A9 were reconstructed into coexpression vectors by PCR amplification of each open reading frame and then subcloned by recombinase reaction. (B) Recombinant protein expression by each plasmid DNA for pET21-S100A8 (Lane 1), pET21-S100A9 (Lane2), pET21-S100A8-S100A9 (Lane 3), and pET21-S100A9-S100A8 (Lane 4) were confirmed by SDS-PAGE. Gels were stained with Coomassie Brilliant Blue (CBB).

Fig. 2

Purification of recombinant S100A8/A9 heterodimer. (A) Soluble fraction of cell lysate (Lane 1) and after nucleic acid precipitation by PEI (Lane 2). Total protein after ammonium sulfate precipitation (Lane 3) and soluble fraction after dialysis (Lane 4). Each lane contained protein sample from 50 μL bacterial cell culture equivalent. (B) Cation-exchange chromatographic purification of S100A8/A9 heterodimer. (C) SDS-PAGE analysis of fractionated samples under reducing (DTT +) and non-reducing (DTT −) conditions. The ratio of S100A9/S100A8 were measured by band intensities using samples electrophoresed under reducing conditions.

Fig. 3

Analysis of protein assembly of S100A8 and S100A9. (A) SEC-HPLC analysis of coexpressed recombinant S100A8/A9 protein (Fig. 2B, Peak1), and individually expressed S100A8 or S100A9 proteins purified by cation-exchange chromatography. (B) SDS-PAGE analysis of purified recombinant S100A8 (Lane 1), S100A9 (Lane 2), and S100A8/A9 heterodimer (Lane 3) under reducing and non-reducing conditions. Analyzed proteins were same as SEC-HPLC injected samples. (C) Thermodynamic diagram of protein assemblies from S100A8 and S100A9 monomers to semi-stable homodimers or stable heterodimers.

Fig. 4

Biological activity of purified S100A8/A9 heterodimer. (A) The invaded cells pass through the protein Matrigel after stimulation by S100A8/A9 heterodimer for 12 h and were counted under microscopic observation. The results represent an average of three independent samples. Data are means±SD. (B) S100A8/A9 stimulated invasive cellular images stained by hematoxylin and eosin. (C) Comparison of biological activity among S100A8 or S100A9 (mixture of monomer and homodimer), S100A8/A9 heterodimers produced in E. coli or HEK293 cells. The assay conditions are same as (A).