Optimized single-step affinity purification with a self-cleaving intein applied to human acidic fibroblast growth factor

To reduce the number of recovery steps during downstream processing and to overcome the limitations of present fusion-based affinity separations, a co ntrollable self-splicing protein element in the form of a mini-intein was u sed to optimize the recovery of proteins for both batch and flow purificati on strategies. The ability to recover purified proteins was demonstrated us ing a tripartite fusion consisting of a maltose binding domain, a truncated intein as a controllable linker molecule, and a protein of interest. To ch aracterize expression level, solubility, cleavage rates, pH and temperature controllability, and protein activity, recombinant human acidic fibroblast growth factor (aFGF) was used as a model protein. A simple mass transport model, based on cleavage reaction-limited mass transfer and constant disper sion, was successfully used to predict product concentration and peak shape in relation to critical process parameters (with no fitting parameters). I nsight into the nature of the cleavage reaction and its regulation was obta ined via temperature- and pH-dependent kinetic data.