CBD Beads
Quantitative Analysis: Crude extract from E. coli containing a plasmid expressing a maltose-binding protein-intein-chitin binding domain fusion protein is passed over a 1 ml column at 4°C. The column is washed with > 10 column volumes of 20 mM HEPES (pH 8.0) 500 mM NaCl, 0.1 mM EDTA, 0.1% Triton-X100. The column is then quickly flushed with 3 column volumes of 30 mM DTT (freshly diluted in cleavage buffer [20 mM HEPES (pH 8.0), 500 mM NaCl, 0.1 mM EDTA]. The flow to the column is stopped, and the column is left at 4°C overnight. MBP is eluted using 3 column volumes of cleavage buffer without DTT.
Regeneration: The packed chitin bead column must be stripped of both the fusion partner (intein-CBD) and the uncleaved fusion protein. Wash the column with 3 bed volumes of the 0.3 M Na0H stripping solution. Allow resin to soak 30 minutes, wash the resin with an additional 7 bed volumes of stripping solution. Wash with 20 bed volumes of water, followed by 5 bed volumes of column buffer.
Use gravity flow to run the column. The column should have a wide diameter.
The column may be regenerated 5 times.
Binding Capacity: 2.0 mg maltose-binding protein/ml bed volume released from the bead after cleavage of the fusion protein expressed from pMYB5.
The used resin should be stored in column buffer.
For long term storage add 0.02% sodium azide to the column buffer.
Stripping the Chitin Column
Uncleaved fusion precursor protein and the intein-tag remain bound to the chitin resin during elution and can be stripped from the resin by 1% SDS or 0.3 M NaOH in column buffer. The elution should be conducted at room temperature to prevent the precipitation of the SDS. Since protein concentrations cannot be determined by the Bradford dye binding assay when SDS is present, the samples should be analyzed by SDS-PAGE.
Regeneration of the Chitin Resin
The chitin resin can be regenerated 4-5 times using the following protocol. Wash with 3 bed volumes of 0.3 M NaOH (stripping solution). Allow the resin to soak for 30 minutes and then wash with an additional 7 bed volumes of Stripping Solution. Rinse with 20 bed volumes of water followed by 5 bed volumes of Column Buffer. The resin can be stored at 4°C. For long term storage 0.02% sodium azide should be added to the Column Buffer.