Cellulose affinity purification of fusion proteins tagged with fungal family 1 cellulose-binding domain.
N- or C-terminal fusions of red-fluorescent protein (RFP) with various fungal cellulose-binding domains (CBDs) belonging to carbohydrate binding module (CBM) family 1 were expressed in a Pichia pastoris expression system, and the resulting fusion proteins were used to examine the feasibility of large-scale affinity purification of CBD-tagged proteins on cellulose columns. We found that RFP fused with CBD from Trichoderma reesei CBHI (CBD(Tr)(CBHI)) was expressed at up to 1.2g/l in the culture filtrate, which could be directly injected into the cellulose column. The fusion protein was tightly adsorbed on the cellulose column in the presence of a sufficient amount of ammonium sulfate and was efficiently eluted with pure water. Bovine serum albumin (BSA) was not captured under these conditions, whereas both BSA and the fusion protein were adsorbed on a phenyl column, indicating that the cellulose column can be used for the purification of not only hydrophilic proteins but also for hydrophobic proteins. Recovery of various fusion proteins exceeded 80%. Our results indicate that protein purification by expression of a target protein as a fusion with a fungal family 1 CBD tag in a yeast expression system, followed by affinity purification on a cellulose column, is simple, effective and easily scalable.
Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Naohisa
N
Sugimoto
Kiyohiko
K
Igarashi
Masahiro
M
Samejima
D000595:Amino Acid Sequence; D044602:Cellulases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D002482:Cellulose / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010843:Pichia; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D000097573:Red Fluorescent Protein
D044602:Cellulases; D002482:Cellulose; D005656:Fungal Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein