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Protein purification by general chromatography techniques
In contrast to affinity-based purification protocols which enables the “fishing out” of the target protein, purification by general chromatography techniques eliminates contaminating proteins using a sequence of different chromatography steps, a protocol.
Here the overall purification power depends primarily on the combination of techniques with independent selectivities. Two-dimensional electrophoresis capitalises on this principle in combining two completely independent protein properties namely isoelectric point and size. In fact, this principle is powerful enough to resolve thousands of proteins in one single two-dimensional electrophoresis experiment!
The standard protocol described below combines the three physical protein properties charge, hydrophobicity and size in terms of the techniques ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC).
In most cases this combination is capable of providing a purity of 90% or better using standard conditions. However, for cases where the purity provided by the standard protocol turns out to be insufficient, advice is given on how to optimise the protocol until the purification goals are met.
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 like cell lysates etc. 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.
Prerequisites
1.Analytical tools
- An off-line detection method specific for the target protein.
This is necessary in order to follow the progress of the purification and to evaluate optimisation measures.
- An off-line detection method to measure total protein content
This is necessary in order to calculate the specific activity of the target protein and to evaluate optimisation measures.
2. Knowledge of the sample stability.
This is necessary in order to choose running conditions compatible with biological activity of the target protein.
! It is of great help to know the isoelectric point (pI), the size and the hydrophobic character of the target protein when designing the individual steps of the purification protocol.
3. Sample condition
The starting material should be free of particulate matter, ionic detergents, and high Mw nucleic acids or columns may be blocked or clogged.
If the starting material contains proteases these should be inactivated or recovery may suffer.
Initial tests
Before deciding on the final purification protocol initial tests are performed on HiTrap columns. Once the final chromatography media and conditions are set, the protocol may be scaled up to handle the amount of sample needed to purify the intended quantity of the target protein.
1. Choice of ion exchanger type and conditions.
HiTrap IEX Test Kit is used to select the appropriate ion exchanger type and to test the suitability of standard IEX conditions.
In order to obtain representative results the column must not be overloaded, still enough of the target protein has to be applied to enable its safe monitoring in the eluate.
The maximum load varies with the type of sample and the conditions used and firm recommendations are not possible. However, as a rule of thumb the HiTrap IEX Kit columns normally accept loads between 5 and 10 mg of total protein.
Buffers
Prepare the following buffers:
- HiTrap Q:
- Buffer A: 20 mM TRIS-HCl, pH 8.0
- Buffer B: 1.0 M NaCl in 20 mM TRIS-HCl, pH 8.0
- HiTrap SP:
- Buffer A: 50 mM acetate, pH 4.5
- Buffer B: 1.0 M NaCl in 50 mM acetate, pH 4.5
Each experiment will require approximately 30ml of buffer A and 20 ml of buffer B.
Sample preparation
- With the recommended maximum sample load in mind, make sure that enough sample is loaded to allow detection of the target protein in the eluate.
- Prior to sample application perform buffer exchange using a PD 10 column to adjust sample conditions to match those of Buffer A.
Experiment
- Run HiTrap Q and HiTrap SP under standard conditions.
- Flow rate: 1ml/min
- Buffers as above
- Gradient: AB in 20 column volumes.
- Fraction size: 1ml.
Evaluation
- Determine the total protein content and the target protein content in all fractions.
- Select the column and conditions that provides the highest relative content of target protein and recovery of biological activity.
! It is advantageous to have the target protein eluting late in the gradient since this allows a larger part of the background to elute in the run-through fraction after adjusting the gradient accordingly.
2. Choice of HIC ligand and conditions.
HiTrap HIC test Kit is used to screen four different HIC chromatography media.
Stability in ammonium sulphate
- Test the sample stability in 0.8 M, 1.0 M, 1.2 M and 1.4 M ammonium sulphate measured as recovery of biological activity.
If the salt interferes with the target-specific assay, perform buffer exchange before assay.
Buffers
Prepare the following eluents:
- Eluent A: 50 mM phosphate pH 7.0 containing the maximum concentration of ammonium sulphate compatible with sample stability.
- Eluent B: 50 mM phosphate pH 7.0.
Each experiment will require approximately 30ml of buffer A and 20 ml of buffer B.
Sample preparation
- With the recommended maximum sample load in mind, make sure that enough sample is loaded to allow detection of the target protein in the eluate.
- Prior to sample application perform buffer exchange using a PD 10 column to adjust sample conditions to match those of Buffer A.
Method
- Run Butyl Sepharose 4 Fast Flow, Octyl Sepharose 4 Fast Flow, Phenyl Sepharose 6 Fast Flow (high sub) and Phenyl Sepharose 6 Fast Flow (low sub) under standard conditions.
- Flow rate: 1ml/min
- Buffers as above
- Gradient: AB in 20column volumes.
- Fraction size: 1ml.
Evaluation
- Determine the total protein content and the target protein content in all fractions.
- Select the column and conditions that provides the highest relative content of target protein and recovery of biological activity.
! It is advantageous to have the target protein eluting late in the gradient since this allows a larger part of the background to elute in the run-through fraction after adjusting the gradient accordingly.
3. Standard protocol
- Combine the active fractions from the selected IEX experiment in 1) and run the HIC step under the conditions selected in 2).
- Determine the specific activity of the target protein in all fractions and calculate the purification achieved.
- Pool active fractions and apply the pool to a gel filtration column.
- Select Superdex 200 HR 10/30 for target protein Mr>50 000.
- Select Superdex 75 HR 10/30 for target protein Mr <50 000.
- Determine the specific activity of the target protein in all fractions, pool the active fractions and calculate the purification achieved.
- Run SDS electrophoresis or MS to determine the purity of the active pool.
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