Characterization of a cellulose binding domain from Clostridium cellulovorans endoglucanase-xylanase D and its use as a fusion partner for soluble protein expression in Escherichia coli.
Different chimeric proteins combining the non-catalytic C-terminal putative cellulose binding domain of Clostridium cellulovorans endoglucanase-xylanase D (EngD) with its proline-threonine rich region PT-linker, PTCBD(EngD), cellulose binding domain of C. cellulovorans cellulose binding protein A, CBD(CbpA), cohesin domains Cip7, Coh6 and CipC1 from different clostridial species and recombinant antibody binding protein LG were constructed, expressed, purified and analyzed. The solubilities of chimeric proteins containing highly soluble domains Cip7, CipC1 and LG were not affected by fusion with PTCBD(EngD). Insoluble domain Coh6 was solubilized when fused with PTCBD(EngD). In contrast, fusion with CBD(CbpA) resulted in only a slight increase in solubility of Coh6 and even decreased solubility of CipC1 greatly. PTCBD(EngD) and Cip7-PTCBD(EngD) were shown to bind regenerated commercial amorphous cellulose Cuprophan. The purity of Cip7-PTCBD(EngD) eluted from Cuprophan was comparable to that purified by conventional ion exchange chromatography. The results demonstrated that PTCBD(EngD) can serve as a bi-functional fusion tag for solubilization of fusion partners and as a domain for the immobilization, enrichment and purification of molecules or cells on regenerated amorphous cellulose.
Xu
Yin
Y
School of Biotechnology & Biomolecular Sciences, University of New South Wales, Sydney, New South Wales 2052, Australia.
Foong
Frances C
FC
D002480:Cellulase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D046974:Clostridium cellulovorans / Q000201:enzymology*; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D011208:Powders; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012995:Solubility
D011208:Powders; D011993:Recombinant Fusion Proteins; D002482:Cellulose; C009865:cuprammonium cellulose; D002480:Cellulase; D043364:Endo-1,4-beta Xylanases