Construction, Expression And Binding Analysis Of The Recombinant Proteins With Double Cellulose-binding Domains (CBDs)

Cellulose-binding domains (CBDs) bind specifically to cellulose, and form distinct domains of most cellulose degrading enzymes.Now,more and more CBDs have been isolated from various cellulose-degrading enzymes,which lack hydrolytic activity. Many researchers have demonstrated that there exists an immense potential for the application of the cellulose-binding domain in the field of biotechnology especially in the field of fiber modification and paper industry.In this study we designed several overlapping primers based on the three kinds of cellulose-degrading enzymes CBD cDNA sequence from volvariella volvacea and constructed three genes including two CBDs by PCR procedure. After reaction of digestion by restrictive enzymes and ligation, the interest genes were cloned into prokaryotic expression vector pet-32a/b, then we got three recombinant expression plasmids: dEG1CBD- pet-32b, dcelc12CBD- pet-32a and dcbd3-101CBD - pet-32a and transfer into E.coli BL21(DE3) to express. Analysis of SDS-PAGE indicated that at 37℃the fusion proteins were expressed as inclusion body and at 30℃, dcbd3-101CBD protein have soluble expression but the other two not. We used Purified and refolded dcelc12CBD, dEG1CBD proteins and dcbd3-101CBD soluble protein to do the binding experiments. The results indicate that all three recombinant proteins can specifically bind to Avicel and the maximal absorption was more than 6μmol/g.The purified proteins were also applied by immersion onto cellulose filter paper and its mechanical properties were tested.The proteins improved the treated paper's tensile strength, bursting strength and Slitting-resistant strength.So, this research demonstrates great potential to develop a novel paper-modifaction reagent.