After identification of sequences of interest from the library, the corresponding genes were synthesized by Eurofins Genomics. Synthesized genes were digested with NcoI-HF and NotI-HF restriction enzymes (New England Biolabs) and ligated into the pET22b+ expression plasmid (Novagen) using T4 DNA ligase. Upon sequence confirmation, the plasmid was transformed into Rosetta(DE3) E. coli (Novagen) to facilitate protein expression.
Protein expression was performed by growth of a 50 mL culture of the above construct in Terrific Broth in the presence of 100 μg/mL ampicillin and 35 μg/mL chloramphenicol overnight at 30°C. The following day, the culture was transferred to 500 mL of fresh broth and grown for 3 h at 30°C. Protein expression was induced with IPTG at a final concentration of 0.5 mM at 30°C for 3 h. Cells were harvested by centrifugation and the protein was harvested from the periplasm by osmotic shock and purified by immobilized metal affinity chromatography and size exclusion chromatography, as described previously [20 (link), 25 (link)]. Gene sequences for Aa, Ac, Ad, and Ca through Cd are available in GenBank under accession numbers KU508539 through KU508545.
Protein expression was performed by growth of a 50 mL culture of the above construct in Terrific Broth in the presence of 100 μg/mL ampicillin and 35 μg/mL chloramphenicol overnight at 30°C. The following day, the culture was transferred to 500 mL of fresh broth and grown for 3 h at 30°C. Protein expression was induced with IPTG at a final concentration of 0.5 mM at 30°C for 3 h. Cells were harvested by centrifugation and the protein was harvested from the periplasm by osmotic shock and purified by immobilized metal affinity chromatography and size exclusion chromatography, as described previously [20 (link), 25 (link)]. Gene sequences for Aa, Ac, Ad, and Ca through Cd are available in GenBank under accession numbers KU508539 through KU508545.
Free full text: Click here
