Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of SpyCEP, a candidate antigen for a vaccine against Streptococcus pyogenes

Abstract

Streptococcus pyogenes (Group A streptococcus; GAS) is an important human pathogen against which an effective vaccine does not yet exist. The S. pyogenes protein SpyCEP (S. pyogenes cell-envelope proteinase) is a surface-exposed subtilisin-like serine protease of 1647 amino acids. In addition to its auto-protease activity, SpyCEP is capable of cleaving interleukin 8 and related chemokines, contributing to GAS immune-evasion strategies. SpyCEP is immunogenic and confers protection in animal models of GAS infections. In order to structurally characterize this promising vaccine candidate, several SpyCEP protein-expression constructs were designed, cloned, produced in Escherichia coli, purified by affinity chromatography and subjected to crystallization trials. Crystals of a selenomethionyl form of a near-full-length SpyCEP ectodomain were obtained. The crystals diffracted X-rays to 3.3 Å resolution and belonged to space group C2, with unit-cell parameters a=139.2, b=120.4, c=104.3 Å, β=111°.

Keywords: GAS; SpyCEP; Streptococcus pyogenes; auto-proteases; vaccine antigens.

Figure 1

(a) Domain architecture of GAS M1 strain SpyCEP (UniProt Q9A180) and the boundaries of the expression constructs prepared: SpyCEP_113–244 (Thr113–Gln244; white), SpyCEP_245–1131 (Ser245–Thr1131; grey), SpyCEP_113–1131 (Thr113–Thr1131) and SpyCEP_34–1613, the full-length ectodomain (Ala34–Leu1614) lacking the C-terminal LPXTG anchor domain (AD, dotted box). The black boxes represent the hexahistidine tags, all of which are at the C-termini. The subtilisin-like protease is composed of two regions spanning residues 124–420 and 572–687. (b) DSC profiles of SpyCEP_245–1131 and SpyCEP_34–1613 showing a clear unfolding transition with similar T _m of 320.5–323 K. (c) The crystal of SpyCEP_34–1613 ectodomain (selenomethionyl form) used for data collection reported here.