Article Figures & Data
Figures
- FIG. 1.
Construction of the pWB980-mini-cbpA1-engB expression plasmid. For B. subtilis expression, we chose a small mini-CbpA, i.e., mini-CbpA1 consisting of a cellulose binding domain, a hydrophilic domain (HLD1), and one cohesin domain (Coh1). Plasmid pWB980, which is a Bacillus expression vector, carries both the constitutively expressed P43 promoter and sacB SP, the engineered levansucrase signal sequence for heterologous protein expression in B. subtilis (23). The sequences coding for the kanamycin resistance marker and levansucrase signal sequence are represented by kmr and sacB SP, respectively. Arrows show the transcription directions for the genes.
- FIG. 2.
Purification procedure for minicellulosomes from B. subtilis WB800 harboring pWB980-mini-cbpA1-engB. SR, superrich; NTA, nitrilotriacetic acid.
- FIG. 3.
Gel filtration chromatography of the water-eluted and Ni2+-nitrilotriacetic acid agarose-purified cellulosome fraction on a Superdex 75 preparation-grade column. Fractions I, II, and III are shown as solid lines, and standards are shown as dotted lines; (a, aldolase [158 kDa]; b, bovine serum albumin [67 kDa]).
- FIG. 4.
SDS-PAGE analysis of fraction II stained with Coomassie blue. Lane 1, fraction II (mini-CbpA1-B and EngB-B); lane 2, purified mini-CbpA1 from B. subtilis WB800 (mini-CbpA1-B); lane 3, crude extract from E. coli harboring the engB gene (EngB-E). Lane Std, molecular weight standards (indicated in thousands).
- FIG. 5.
Characterization of fractions I and II. (A) Western blot showing detection with antisera raised against HLD1 and EngB, respectively. Anti-HLD1 was made against the hydrophilic domain 1 of CbpA, and anti-EngB was made against EngB. (B) Zymogram analysis with 0.1% carboxymethyl cellulase (CMCase) incorporated into the polyacrylamide. Lane Frc I, fraction I; lane Frc II, fraction II. Molecular weights are indicated on the left in thousands.
