A Novel Protein Purification Approach Using Elastin-Like Polypeptides (ELP) With His-Tag Assistance.

Abstract

Protein purification is essential for drug development, antibody production, and structural biology. We propose a cost-effective chromatography method using elastin-like polypeptide (ELP) as an aggregating core. In this approach, a chilled (target protein)-ELP fusion is loaded onto an immobilized metal affinity chromatography (IMAC) column equilibrated with low-salt buffer. Impurities are removed with warm high-salt buffer washes. Warming the column above the ELP's transition temperature (T_m) triggers ELP aggregation, physically trapping the target protein between beads. Subsequent stringent washing (high salt/imidazole) eliminates residual contaminants. Finally, cooling with cold low-salt buffer reverses aggregation, eluting the purified target protein. This method eliminates the need for advanced chromatography systems while achieving high purity through dual mechanisms: (1) IMAC affinity and (2) temperature-dependent physical capture. The ELP's reversible phase transition offers a simplified yet efficient purification platform, particularly valuable for lab-scale production of challenging proteins. Key features • This protocol requires an elastin-like polypeptide tag at the C-terminus of the target protein. • This protocol requires a His-Tag at the N-terminus of the target protein. • This protocol requires the use of colored/chromogenic proteins to enable real-time visual monitoring of chromatographic progression. • This protocol yields a highly pure protein by manually operating a Ni-bound resin at two different temperatures.