An efficient system for small protein expression and refolding

Abstract

The low expression yield and poor refolding efficiency of small recombinant proteins expressed in Escherichia coli have continued to hinder the large-scale purification of such proteins for structural and biological investigations. A system based on a small fusion partner, the B1 domain of Streptococcal protein G (GB1), was utilized to overcome this problem. We have tested this system on a small cysteine-rich toxin, mutant myotoxin alpha (MyoP20G). The highly expressed fusion protein was refolded using an unfolding/refolding protocol. Due to the small size of GB1, we were able to monitor the unfolding/refolding status by heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The final product yielded well-resolved NMR spectra, with a topology corresponding to the natural product. We conclude that GB1 not only increases the expression level but also enhances the refolding of small proteins.

Fig. 1

Construction of expression vector, pGBO, for expression of MyoP20G. Three restriction endonuclease sites are shown in (A). In the final fusion protein sequence (B), the linker and the thrombin recognition site are revealed by italic characters, with a star denoting the cleavage site.

Fig. 2

Spectra of samples from different stages of the folding process. (A) ¹H–¹⁵N HSQC spectrum of the fusion protein under unfolding conditions. Sample was in 25 mM Tris buffer, pH 7.5, and 5 mM DTT. The majority of the poorly dispersed signals, which are encircled by a dashed ellipse, correspond to backbone NH resonances of unfolded MyoP20G. (B) ¹H–¹⁵N HSQC of fusion protein during the refolding process. Sample was in refolding buffer solution (see Materials and methods). Arrows indicate signals from NH resonances associated with the folded state of MyoP20G. (C) ¹H–¹⁵N HSQC of MyoP20G after digestion with protease to remove GB1 and purification. Sample was in 25 mM Tris buffer, pH 7.5, and 460 mM NaCl. (D) ¹H–¹⁵N HSQC of MyoP20G in 25 mM sodium acetate–acetic acid buffer, pH 5.4.

Fig. 3

Cation-exchange chromatography profile of refolded MyoP20G. (A) Crude refolding product, (B) purified refolded sample. The narrow peak in (A) corresponding to the correctly folded MyoP20G is labeled by an arrow.