CBM21 starch-binding domain: a new purification tag for recombinant protein engineering.
The use of protein fusion tag technology simplifies and facilitates purification of recombinant proteins. In this article, we have found that the starch-binding domain derived from Rhizopus oryzae glucoamylase (RoSBD), a member of carbohydrate-binding module family 21 (CBM21) with raw starch-binding activity, is favorable to be applied as an affinity tag for fusion protein engineering and purification in Escherichia coli and Pichia pastoris systems. To determine suitable spatial arrangement of RoSBD as a fusion handle, enhanced green fluorescent protein (eGFP) was fused to either the N- or C-terminus of the SBD, expressed by E. coli, and purified for yield assessment and functional analysis. Binding assays showed that the ligand-binding capacity was fully retained when the RoSBD was engineered at either the N-terminal or the C-terminal end. Similar results have been obtained with the RoSBD-conjugated phytase secreted by P. pastoris. The effective adsorption onto raw starch and low cost of starch make RoSBD practically applicable in terms of development of a new affinity fusion tag for recombinant protein engineering in an economic manner.
Lin
Shu-Chuan
SC
Institute of Molecular and Cellular Biology, Department of Life Science, National Tsing Hua University, Hsinchu 300, Taiwan, ROC.
Lin
I-Ping
IP
Chou
Wei-I
WI
Hsieh
Chen-An
CA
Liu
Shi-Hwei
SH
Huang
Rong-Yuan
RY
Sheu
Chia-Chin
CC
Chang
Margaret Dah-Tsyr
MD
D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D012233:Rhizopus / Q000201:enzymology; D013213:Starch / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D013213:Starch; D005087:Glucan 1,4-alpha-Glucosidase