chapter  39
4 Pages

Recombinant Proteins

Principle The gene coding for the target protein X is cloned in a vector of expression. This vector is a plasmid with a bacterial promoter (most often inducible) followed by part of the sequence coding for a bacterial protein, of a sequence corresponding to a cleavage site recognized by a protease and a polycloning site at the level of which the target gene will be introduced, respecting the reading frame for translation into amino acids. After transformation, the bacteria that have integrated a recombinant vector are selected on a selection medium, multiplied, then harvested and lysed so as to obtain a raw cellular extract containing all the proteins synthesized, including the fusion protein. From this raw extract, the fusion protein is purified by affinity chromatography on column: groups, called ligands, are fixed on the column and interact specifically with the fusion protein, retaining the latter, while the other proteins are eliminated. The fusion protein is recovered after specific elution and a cleavage is induced between the bacterial protein and protein X by incubation in the presence of a suitable protease.-A second affinity chromatography can be carried out to eliminate the bacterial protein: only the latter is retained on the column, while protein X is eluted.