Development of recombinant proteins for selection, immobilization and expansion of stem cells

Cellulose binding domains (CBDs) are found in cellulolytic microorganisms as discrete domains in free cellulases or as cellulosomes, which are extracellular multi-enzyme complexes. CBDs bind to cellulose and help the catalytic domains to access cellulose substrates. CBDs are used as affinity tags for immobilizing cells, proteins or molecules on cellulose matrices. They can also be used in protein engineering to alter protein expression and solubilities. Cohesins and dockerins are domains exclusive to cellulosomes. They interact with high affinity and the interactions are Ca2+-dependent. Chelation of Ca2+ causes irreversible conformational change to dockerins thus disrupting the interactions. The first aim of this project was to validate a putative CBD from endoglucanase EngD of C. cellulovorans, to test its effect on solubility as a fusion in chimeras. The second aim was to use chimeric proteins containing CBD, cohesin, dockerin and LG to establish a system for efficient cell immobilization, expansion and harvest in hollow cellulose fibres. A putative CBD from an enzyme with its natural linker (PTCBDengD), a CBD from a scaffoldin (CBDcbpA), three cohesin domains from different strains (Cip7, Coh6 and CipC1) and an antibody binding protein (LG) were used to construct various chimeric and fusion proteins. The two CBDs were fused to different cohesins and LG respectively and the chimeras? solubility was analyzed. The results showed that fusing with CBDcbpA did not significantly help to increase the solubility of the insoluble domain Coh6 and it even greatly reduced the solubility of the soluble domain CipC1. In contrast, PTCBDengD fusion increased the solubility of Coh6 by three fold and it did not alter the solubility of soluble protein/ domains. These results suggested that PTCBDengD may be a better domain to use as a fusion tag for expression and other biotechnology applications. Cellulose binding specificity of PTCBDengD and its chimeric proteins were tested and SDS-PAGE analysis results clearly demonstrated that PTCBDengD and its chimeras specifically bound to crystalline cellulose Avicel and non-crystalline cellulose Cuprophan. The results confirmed that PTCBDengD is a true CBD. Chimeric protein pairs CBDcbpA-Cip7/ LG-Doc and Cip7-PTCBDengD/ LG-Doc were used to build the scaffold on Cuprophan hollow cellulose fibre for reversible cell immobilization studies. Cell adhesion assay results showed that the double-chimera systems efficiently immobilize cells onto Cuprophan hollow fibre. Dissociation of LG-Doc from amorphous cellulose Cuprophan-bound CBDcbpA-Cip7 by EDTA treatment resulted in decrease of cell binding by almost 90%; however, re-association of LG-Doc after EDTA dissociation only achieved 50% efficiency of cell immobilization. Dot blot and SDS-PAGE analysis showed that dissociation/ re-association of LG-Doc to Cip7-PTCBDengD could be decreased in was interfered by the presence of cellulose. Preliminary results indicated that crystalline cellulose Avicel may improve dissociation/ re-association efficiency.In conclusion, studies on recombinant proteins validated CBDengD's specific affinity to cellulose and its solubilizing effect on its fusion partner. Chimera pairs CBDcbpA-Cip7/ LG-Doc and Cip7-PTCBDengD/ LG-Doc are effective in cell immobilization. However, optimization is required to develop recyclable protein scaffolding and complexes on cellulosic matrices