Order of steps in enzyme purification

Usually a purification has several steps. 1) 2) Preparation of crude extract (cell lysis; removal of cell walls/membranes in case of soluble proteins) (Optional: removal of nucleic acids, ribosomes with protamine sulfate)

3a) (NH4)2SO4 precipitation; protein to be purified should remain just soluble 4a) Hydrophobic interaction chromatography: application at high salt, elution by decreasing salt gradient 5a) IEC (eventually desalting before application; elution with increasing salt) OR: 3b) (NH4)2SO4 fractionation; dialysis; 4b) IEC 5b) Hydrophobic interaction IEC and hydrophobic interaction chromatography are usually the first column steps during a purification.

6) Gel filtration and/or hydroxyapatite chromatography Gel filtration and HAP are usually applied as later column steps in purification. If applicable, IMAC or bio-affinity chromatography may replace steps 4 and/or 5 and/or 6. In practice, IMAC and bio-affinity chromatography are often preceeded by IEC: removal of impurities by IEC might increase the lifetime of the expensive resins applied in IMAC and bio-affinity chromatography. Purification to homogeneity of a protein requires often 5 10 steps (including desalting and concentration steps). Yield is important. If, in an 8-step purification, the yield for each step is 70%, the final yield after 8 steps is only 5% ! Nevertheless, sometimes a compromise should be made between yield and purity. Recombinant DNA techniques allow overproduction of proteins. Overproduction of proteins may dramatically increase the amount of protein to be purified in a crude extract. Purification of a protein is much easier when the protein is already present in high amounts in the crude extract !!