CBM1 Domain Fusion Protein Production

The CBM1 constructs were designed to have a carrier protein fused to the CBM1 domain by a flexible linker, in addition to the N-terminal pelB signal peptide and a 6His purification tag on the C-terminus. Alkaline Phosphatase (AP) was chosen as carrier protein since it was known to produce well and could be used in colorimetric assays. The pelB, linker, and CBM1 sequences were ordered as GeneArt Strings (ThermoFisher) and the AP segment was obtained with PCR (using KAPA HiFi DNA Polymerase from KAPA Biosystems and primers from Eurofins), all flanked with BsaI restriction sites and appropriate 4bp overlaps. Using the Golden Gate cloning method [46 (link)] the pieces were combined (digest with BsaI-HF from NEB) and ligated (T4 DNA Ligase NEB) into a pET28a (+) expression vector and transformed in chemically competent TOP10 E. coli cells. The resulting plasmids were obtained by miniprep (NucleoSpin Plasmid from Macherey-Nagel). The resulting constructs were verified by 1% agarose gel electrophoresis and sequencing (FIMM, Helsinki).