Premium
Molecular Design and functional analysis of Starch Binding Domain of Rizopus oryzae Glucoamylase
Author(s) -
Ng Sim-Kun,
Fang Tsai-Wei,
Lee Yuan-Chuan,
Huang Han-Tsz,
Jiang Ting-Ying,
Ci Yuan-Pei,
Chou Wei-I,
Chang Dah-Tsyr Margaret
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.lb232
Subject(s) - isothermal titration calorimetry , chemistry , trimer , rhizopus oryzae , biochemistry , recombinant dna , carbohydrate binding module , starch , glycan , binding domain , dimer , binding site , gene , glycoprotein , glycoside hydrolase , organic chemistry , fermentation
Starch‐binding domain (SBD) is a functional domain which effectively adsorbs onto granular starch and other soluble oligosaccharides. The 11.7 kDa N ‐terminal SBD of Rhizopus oryzae glucoamylase ( Ro SBD) belongs to carbohydrate binding module (CBM) family 21. Ro SBD has been developed as a purification tag that successfully overexpress and purify recombinant proteins in bacteria and yeast. Here Ro SBD‐eGFP expression in mammalian cell line HEK293FT is also successfully expressed, and recovered with high purity (95%) and fair yield (30 mg/L). Since tandem repeat of CBMs is known to promote ligand binding affinity, multivalent protein binders including mouse IgG1 Fc and collagen‐like peptide (CLP) are engineered to generate respectively dimeric and trimeric Ro SBD. The polymeric Ro SBDs are expressed stably with high quantity in E. coli and form desired dimer and trimer. Improvement in binding activity of polymeric Ro SBDs to granular starch and soluble oligosaccharides is observed employing depletion isotherm and isothermal titration calorimetry (ITC). Although natural CBM‐glycan interaction is usually too weak to be precisely detected, the improvement of binding affinity between Ro SBD and several glycans may enable us to investigate novel binding partners of Ro SBD. These results may also facilitate development of novel protein purification systems vitally important for biotechnological applications.