Expression, purification and structural characterization of the scaffoldin hydrophilic X-module from the cellulosome of Clostridium thermocellum

• Published in: Protein expression and purification Vol. 38; no. 2; pp. 258 - 263
• Main Authors: Adams, Jarrett J., Jang, Christopher J., Spencer, Holly L., Elliott, Marc, Smith, Steven P.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-12-2004
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Cellulase - chemistry; Cellulose degradation; Cellulosome; Cloning, Molecular; Clostridium - enzymology; Clostridium thermocellum; Gene Expression Regulation, Enzymologic; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - isolation & purification; Molecular Sequence Data; Multienzyme Complexes - chemistry; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - isolation & purification; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Sequence Homology, Amino Acid; X-module; Cellulosome; X-module; Clostridium thermocellum; Cellulose degradation
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2004.08.018

The cellulosome is a membrane-bound, extracellular multi-subunit complex responsible for the degradation of crystalline cellulose by a number of organisms including anaerobic bacteria and fungi. The hydrophilic X-module (CipA-X) from the modular scaffoldin subunit of
Clostridium thermocellum cellulosome has been proposed to play various roles in cellulosomal function, including thermal and structural stability. Towards elucidating the function of CipA-X using structural and biophysical studies, the region comprising residues 1692–1785 from the
C. thermocellum CipA cDNA encoding CipA-X was cloned into a pET21b expression vector. When expressed in
Escherichia coli, the C-terminal His-tagged protein accumulated in the insoluble fraction. Cell fractionation experiments showed that the recombinant protein was localized to inclusion bodies. Refolding and purification involved denaturation of the whole cell lysate by addition of urea, followed by a nickel-Sepharose chromatography step and dialysis into native conditions (25
mM Tris–HCl, pH 7.4, 50
mM NaCl, and 10
mM EDTA). A final gel filtration step purified the protein to homogeneity, yielding 40
mg/L. The two-dimensional
1H–
15N correlation spectrum of uniformly
15N-labelled CipA-X showed the characteristics of a well-folded protein comprising significant β-structure, which is in agreement with the circular dichroism data.