Observations and practical tips on metal affinity chromatography and protein refolding techniques

Immobilized metal affinity chromatography (IMAC) is a specific high‐capacity technique used in large‐scale purification of proteins. In spite of the robustness of the technique, several factors still affect column binding capacity, yield and purity of proteins during IMAC. Using (His) 6 ‐tagged proteins with comparably high levels of expression, we investigated the effect of protein size, flow‐rate, number and position of (His) 6 tag on protein yield using commercial Ni 2+ IMAC columns. Results suggest that regardless of manipulation of flow rate, (His) 6 tag placement and number, size of protein is the major factor affecting binding and yield during IMAC purification. Small and medium sized proteins (<50 kDa) bind more tightly to the column than bigger proteins, resulting in higher yield during purification. In another study, we employed a modified gradient gel filtration technique to successfully refold a spectrum of highly aggregation‐prone enzymatic proteins back to their native states during denaturing purification. Some of the proteins chosen are well‐known to precipitate significantly during refolding. However, using this technique, precipitation was either so minimal or non‐existent. Enzymatic assays and dynamic molecular mass determination confirmed that the proteins were correctly refolded and biologically active. This strategy provides a robust approach to refolding denatured proteins that can also be used simultaneously as buffer exchange and additional purification step. Support or Funding Information The Welch Foundation