Protein purification strategies

In contrast to affinity-based purification protocols which, enable the “fishing out” of the target protein, purification by general chromatography techniques eliminates contaminating proteins using a sequence of different chromatography steps.

Here the overall purification power depends primarily on the combination of techniques with independent selectivities. Two-dimensional electrophoresis capitalizes on this principle by combining two completely independent protein properties, namely isoelectric point and size. In fact, this principle is powerful enough to resolve thousands of proteins in a single two-dimensional electrophoresis experiment!

Protein purification protocols combining the three physical protein properties - charge, hydrophobicity and size are in many cases capable of providing a purity of 90% or better using standard conditions.

The complete protocol is divided into three phases with separate objectives:

Capture, Intermediate purification, Polishing (CIPP)

The protocol is designed to handle quite complex and sometimes rather dilute starting materials such as cell lysates and the objective of the capture phase is to reduce the amount of background proteins and to concentrate the target protein.

The intermediate purification phase aims at removing most of the contaminant more closely related to the target protein.

Finally the polishing phase serves to eliminate any traces of specified contaminants and aberrant forms of the target protein (polymers, subunits etc.) that may have been formed during isolation and purification.

Developing purification protocols

The development of a purification protocol involves four basic phases:

1. Forming a platform for the development
The platform should include:
Intended quantity and quality of the end product.
Available information on the physical properties of the target protein.
Available information on the type of starting material and content of the target protein.
Information on the range of conditions compatible with sample stability (the sample stability window, SSW).

2. Running initial purification experiments.
The Standard Purification Protocol is used as a starting point for the development. In many cases, this will provide sufficient purity, while in others it may need to be further optimized.

3. Optimizing the purification protocol.
Guided by the outcome of the standard protocol experiment, the purification steps are optimized and/or recombined until satisfactory results are obtained.

4. Adapting the scale of the final purification protocol.
Normally it is practical to run initial and optimization experiments on small columns in HiTrap™ format. If enough purified material cannot be obtained with HiTrap columns , the protocol is scaled up according to certain simple rules.

How to combine purification steps

The purification of crude biological samples can be divided into three distinct phases (Fig 1).


Fig 1. Preparation and CiPP. The three-stage strategy applied when purifying crude samples.

The capture (C) step aims at concentrating the sample and removing the bulk of the contaminants. Emphasis is primarily on speed and load capacity. Media with high load capacities and good flow properties are often used under step elution conditions.
The intermediate purification (iP) step(s) aims at separating the components of the now concentrated, partially purified sample. Emphasis here is on maximum resolution since the remaining contaminants have rather closely related chromatographic properties. This is done by combining techniques of independent selectivities run in high resolution mode (gradient elution, smaller bead, sizes etc.).
Flow rates and load capacities have to be restricted or resolution will suffer.
The polishing (P) step(s) serves to achieve the final purity and to eliminate contaminants such as polymers and is also often used to condition the final product, (e.g. to remove salts, when the final product is to be lyophilised).

No chromatographic technique will provide 100% yield of active material and the overall yield will therefore depend on the number of steps included in the purification protocol.
As seen in figure 2, even a reasonable yield of 80% per step, results in only 20% overall yield after 8 purification steps.

Fig 2. Overall yields at different levels of yield/step.

Minimizing the number of steps (including inter-step conditioning) is therefore important to arrive at a high
overall yield.

To minimize the number of steps, method development should aim at optimizing each step for the intended purpose and arranging them in an order that minimizes inter-step treatments (Fig 3)


Fig 3 Start conditions and end conditions for commonly
used chromatography techniques.


Purification by removing the target molecule from the contaminants.
Affinity chromatography techniques are very specific for the target molecule or for a group of molecules with closely related biological properties. This makes them capable of
“fishing out” the target molecule (or the group), leaving all contaminants behind.
When applicable, these techniques are preferred, since they drastically simplify the purification protocol.

Purification by removing the contaminants from the target molecule.
When a suitable affinity chromatography technique is not at hand, one has to rely on a sequence of general chromatography techniques to remove the contaminants.
When nothing is known about the target protein, a typical purification protocol employs the IEX- HIC- GF sequence of purification steps.

The different techniques used in purification are not suitable for all stages of any given three-phase strategy. Guidance on selection of appropriate three-phase strategies is given below.



Suitability of purification techniques for CIPP

Handbook

Protein Purification Handbook pdf

Protein Purifier™ Software

Protein Purifier™ Software is a software-based learning aid that simulates the experience of working through protein purification problems using a range of separation and analytical techniques. It gives a practical insight into strategies commonly employed in purification schemes without wasting time and materials. Protein Purifier Software Free Download (zip-file)