Expression, purification and structural characterization of the scaffoldin hydrophilic X-module from the cellulosome of Clostridium thermocellum.
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 beta-structure, which is in agreement with the circular dichroism data.
Adams
Jarrett J
JJ
Department of Biochemistry, Queen's University, Kingston, Ont., Canada.
Jang
Christopher J
CJ
Spencer
Holly L
HL
Elliott
Marc
M
Smith
Steven P
SP
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002480:Cellulase / Q000737:chemistry*; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; C080369:CipA protein, Clostridium; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C473132:cellulosome, Clostridium; D002480:Cellulase