Characterization of the CipA scaffolding protein and in vivo production of a minicellulosome in Clostridium acetobutylicum.
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.
Sabathé
Fabrice
F
Centre de Bioingénierie Gilbert Durand, UMR-CNRS 5504, Lab. Ass. INRA, Institut National des Sciences Appliquées, 135 avenue de Rangueil, 31077 Toulouse, France.
Soucaille
Philippe
P
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002480:Cellulase / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D003013:Clostridium / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*
D001426:Bacterial Proteins; C080369:CipA protein, Clostridium; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D002482:Cellulose; D002480:Cellulase