Performing an Isolation of Native Complexes

Natural multiprotein complexes (non-recombinant, without affinity tags) can be purified for structural and functional studies using classical biochemical methods. The natural source of the target complex is used as starting material for the purification and a combination of chromatography and other techniques is used to isolate the complex. This approach, in principle, is identical to that which has been used successfully for the isolation of individual proteins since the 1960s.

Some points to consider with this approach are listed in Table 1.

Table 1.Points to consider for the isolation of native complexes using a classical biochemical approach.

Pros	Cons
Use of the relevant biological source limits the risk for artifacts	Large amounts of starting material are required for the isolation of naturally low abundant complexes
No manipulation (e.g., affinity tagging) of the subunits limits the risk for artifacts	A new purification procedure has to be empirically developed for every complex. Purification schemes tend to have multiple chromatographic steps and be more tedious than for tagged proteins
No re-assembly of the complex required – limits the risk for artifacts

A purification scheme is best designed by combining orthogonal techniques in sequence, for example, combining affinity chromatography (separation according to biospecificity) with ion exchange (separation according to charge properties) and gel filtration (separation according to size). Different techniques should be combined in a fashion that minimizes the requirements for sample treatment between the purification steps (see Principles and standard conditions for different purification techniques). Useful chromatography techniques are shown in Table 2.

Table 2.Chromatography techniques and considerations for multiprotein complex purification.

Technique	Separation principle	Pros (+) and cons (-) for multiprotein complex purification
Gel filtration	Size and shape	+ Mild separation conditions; separation can be achieved over a broad pH and ionic strength range + Additives can be used
Affinity chromatography	Biospecific affinity	+ Excellent specificity - Affinity ligands (for the chromatography beads) cannot be found for all proteins - Elution may require conditions (pH, ionic strength) that may disrupt intracomplex interactions
Ion exchange chromatography	Charge	+ High resolution can often be achieved - A salt gradient is often used for elution This may disrupt intracomplex interactions
Hydrophobic interaction chromatography	Hydrophobicity	+ A useful complement to the other techniques - High salt concentrations (e.g., 1–2 M ammonium sulfate) are often used during sample application. This may disrupt intracomplex interactions

It is important to keep the number of steps to a minimum to maximize purification yields. However, for challenging purifications, such as those for multiprotein complexes, a larger number of purification steps may have to be employed to achieve the desired purity. For instance, the human (20S) proteasome (containing 14 subunits, Mr ~700 000) and precursor complexes were isolated from a human cell line using a combination of anion exchange chromatography, sucrose gradient centrifugation, affinity chromatography, hydrophobic interaction chromatography, and gel filtration.

Harsh separation conditions involving extremes of pH or ionic strength should be avoided in complex purification in order to minimize the risk for dissociation of subunits during the purification process.

Gel filtration separates according to size and is an ideal technique for isolation of multiprotein complexes because it is mild, and also because many complexes are much larger than the main contaminants. Useful gel filtration media for protein complexes and large molecules are listed in Table 3.

Table 3.Gel filtration media with high porosity and thus particularly suitable for multiprotein complex purification.

Product	Useful separation range*	Comment
Superdex 200	10 000 – 600 000	Available as prepacked columns
Superose™ 6	5 000-5 000 000	Available as prepacked columns
Sephacryl™ S-300 HR	10 000 – 1 500 000	Available as prepacked columns
Sephacryl S-400 HR	20 000 – 8 000 000
Sephacryl S-500 HR	No data (higher porosity than Sephacryl S-400 HR)	Exclusion limit ~200 nm
Sephacryl S-1000 SF	No data (higher porosity than Sephacryl S-500 HR)	Exclusion limit ~400 nm

*Mr for globular proteins

Materials

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