Characterization of the CipA scaffolding protein and in vivo production of a minicellulosome in Clostridium acetobutylicum

Abstract

The cipA gene encoding the Clostridium acetobutylicum scaffolding protein CipA was cloned and expressed in Escherichia coli. CipA contains an N-terminal signal peptide, a family 3a cellulose-binding domain (CBD), five type I cohesin domains, and six hydrophilic domains. The uniqueness of CipA lies in the enchainment of cohesin domains that are all separated by a hydrophilic domain. Affinity-purified CipA was used in equilibrium-binding experiments to characterize the interaction of CipA with crystalline cellulose. A K(d) of 0.038 micro M and a [C](max) of 0.43 micro mol of CipA bound per g of Avicel were determined. A mini-CipA polypeptide consisting of a CBD3a and two cohesin domains was overexpressed in C. acetobutylicum, yielding the in vivo formation of a minicellulosome. This is to our knowledge the first demonstration of the in vivo assembly of a recombinant minicellulosome.

FIG. 1.

(A) Schematic diagram of the CipA scaffolding protein from C. acetobutylicum. CipA contains 1,845 amino acids and has a molecular mass of 154 kDa. (B) Structure-based sequence alignment of family 3a CBDs. Secondary-structure elements are indicated and enumerated. Proposed cellulose-binding residues are bpxed, as are the homologous residues in the other CBDs. Calcium-binding residues are shown on a dark shaded background. Highly conserved residues, which occupy the shallow groove of unknown function on the top face of the molecule, are shown on a solid background.

FIG. 2.

Purification of CipA protein. Lanes: 1, molecular mass markers; 2, purified CipA fraction. Arrow indicates full-length CipA scaffolding protein. Arrowheads show the bands corresponding to CipA degradation products.

FIG. 3.

Double-reciprocal plot to quantify the parameters of adsorption of CipA to Avicel.

FIG. 4.

Cellulosome characterization. Immunological detection of cellulolytic complex purified by native PAGE from wild-type C. acetobutylicum (A) and a recombinant strain transformed with the prSCipA plasmid (B). Detection was done with the anti-Coh5 antiserum. (C) SDS-PAGE analysis of the minicellulosome. Mini-CipA and Cel48A were identified with anti-Coh5 and C. cellulolyticum Cel48F antisera, respectively.