Extending the cellulosome paradigm: the modular Clostridium thermocellum cellulosomal serpin PinA is a broad-spectrum inhibitor of subtilisin-like proteases

Abstract

Clostridium thermocellum encodes a cellulosomal, modular, and thermostable serine protease inhibitor (serpin), PinA. PinA stability but not inhibitory activity is affected by the Fn(III) and Doc(I) domains, and PinA is a broad inhibitor of subtilisin-like proteases and may play a key role in protecting the cellulosome from protease attack.

Fig 1

(A) Schematic representation of the modular structure of PinA with the rPinA75 and the rPinA614 constructs as indicated. Briefly, pinA was PCR amplified from pET21-SK2 (2) and cloned into pET-28c(+) (Novagen), placing the gene under the control of the vector-borne T7 promoter and facilitating the fusion of a C-terminal 6×His tag. The final rPinA75- and rPinA614-expressing plasmid constructs were confirmed by Sanger sequencing. The PCR primers used facilitated the addition of an N-terminally located Strep II tag and a factor Xa cleavage site to the serpin and a C-terminally located factor Xa cleavage site. The signal sequence (gray), Strep II tag/factor Xa site (Strep II/Xa), factor Xa site/6×His tag (Xa/6XHis), fibronectin III (Fn III), dockerin (Doc I), and serpin regions are indicated. (B) SDS-PAGE analysis of rPinA75 (1) and rPinA614 (2).

Fig 2

(A) Native PAGE analysis of recombinant rPinA614 harvested at 4, 5, 6, 7, 8, 9, and 24 h. The metastable (rPinA614) and latent (rPinA614-L) forms are indicated. The identity of rPinA614 and rPinA614-L from 5 to 24 h was confirmed by LC-EIS MS/MS. (B) SDS-PAGE analysis of recombinant rPinA614 harvested at 5, 6, 7, 8, 9, and 24 h. (C) Native PAGE analysis of the thermostability profile of rPinA614 heated at 70°C and harvested as indicated. The identity of rPinA614 and rPinA614-L at all time points was confirmed by LC-EIS MS/MS with the exception of rPinA614-L at 60 min. (D) Thermostability analysis of rPinA75 and rPinA614. The thermal stability of rPinA was determined essentially as described by Kang et al. (2) except that the reactions were performed at 60°C. The thermostability analyses were independently performed at least twice per sample.