Azido-PEG3-NHS ester

Wang, Xue. Diss. University of Georgia, 2020.

Here is a simple and general method to modify cellulase/CBM with DNA linkers. Cellulase and CBM were first coupled to lysine residues via azido-PEG3-NHS-ester to obtain azide functional groups. Protein-N3 then functionalized DNA (DNA-DBCO) with dibenzocyclooctyne (DBCO) to tether the single strands.
Azide modification of cellulase/CBM (cellulase/CBM-N3). 200μg/ml cellulase and CBM solutions were prepared in 100mM sodium phosphate buffer containing 100mM NaCl (pH=7). Azide-PEG3-NHS ester was dissolved in dimethyl sulfoxide (DMSO) at 100mg/ml and added to the cellulase/CBM solution at a molar ratio of 1000:1 to protein and incubated overnight. The ester was removed by Zeba™ spin desalting columns. Protein concentration was confirmed by BCA assay. Cellulase/CBM conjugated to DNA linker (cellulase/CBM-DNA). The DNA linker was dissolved in 100mM sodium phosphate buffer containing 100mM NaCl (pH=7) and mixed with cellulase/CBM-N3 solution at a molar ratio of 5:1. The sequence of DNA-DBCO is shown in the purification of unconjugated DNA-DBCO.