Development of purification protocol for soluble over expressed protein

- Protein crystallography

Main steps:

   - Protein purification
     - Introduction
     - Step-by-Step
     - Common sense
       - Common sense strategy
       - Purification protocol
       - Purification of soluble protein
       - Purification of DNA-binding protein
       - Timing of purification
     - Protocols
     - Charts & Tables
     - Appendix
   - Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

Protocols and tips in protein purification or How to purify protein in one day.

Preparations

Prepare stock solutions, columns and chromatographic systems.
Using ExPasy data base find information on the Target Protein. Using ProtParam Tool produce a primary structure analysis. Work out MW, amino acid composition, pI and extinction coefficient of the protein
If the protein has Cysteines, you may consider adding 1mM DTT to buffers during the purification.
Perform a solubility test on the cell paste you are going to use to confirm that Target Protein was expressed as a soluble protein (see Solubility test protocol in Protocols section). If during this test you have revealed that Target Protein reversibly precipitates in low salt conditions, go to Special purification protocols section.
Prepare Crude Extract in buffer A (50mM tris-HCl pH 8.0) (see Crude extract preparation protocol in Protocols section). For our scale the optimum amount of total protein in Crude Extract is 100-500 mg.

Anion exchange chromatography

Target Protein

A Target Protein with pI below 6.5 should be bound to the column, nevertheless, collect flow though fraction in the separate container, do not discard it. Elute proteins with 300 ml of a linear gradient of NaCl from 0 to 0.5M in buffer A. Collect 8 ml fractions. If Target Protein has activity which can be easily assayed (time required for assay is shorter then time required for SDS-PAGE analysis) go to step 7, if assay is not possible go to step 8.
Analysis for activity. Analyse every third/fourth fraction for activity. When a fraction with activity is found, assay each fraction around the active fraction to find every fraction which is active. Check the protein concentration in those fractions using Bradford method and calculate specific activity. If you know the specific activity of the pure protein, calculate the purity of the Target Protein in the active fractions. Separately combine fractions with purity over 60%, and fractions with purity 20%-60%. If you have no fractions with purity over 50% or if you do not know the specific activity of the pure protein then ignore fractions with the lowest specific activity (containing 10-20% of all activity) combine the 2-3 fractions with the highest specific activity, taking about 60-70% of all activity - Peak and separately combine the rest of the active fractions - Sides. If purity of peak is higher than 60% go to step 10, if it is lower or unknown, go to step 11.
Analysis of protein concentration. Analyse protein concentration in every second fraction. If expression of Target Protein was higher than 15%, you will probably find a very distinct protein peak, normally in 4-6 fractions. Take 2-4 Peak fractions separately and 2-4 fractions from both sides of the peak separately (Sides). Go to step 10. If you are not sure or you can not find a sharp protein peak, or if the Target Protein level in crude extract was lower than 10%, go to step 9.
SDS-PAGE analysis. You will need to analyse fractions using SDS-PAGE. Only fractions containing protein should be analysed, including the flow through fraction. It is not necessary to check each fraction, every second fraction is enough. MW markers should be represented on the gel and so should a crude extract (Crude Extract ) sample. Take 20µg of Crude Extract . From the fractions take equal volumes so that protein in each sample remains between 2 and 25µg. For example, the highest protein concentration in fractions was 1 mg/ml, so take 20-25µl samples from fractions with protein concentration >= 0.1 mg/ml. Normally in this step there are about 25-30 fractions containing any significant amount of protein, so a 15 well gel normally suits the analysis. After the gel run is completed, stain gel for 5 minutes with fresh stain and destain for about 10 minutes. Analyse gel to find fractions with the Target Protein. Combine 2-4 fractions with the highest content of the Target Protein (Peak) and combine 2-4 fractions from the sides of the peak (Sides) (so you have a Peak and a Sides fraction). Check volume and protein concentration in both Peak and Sides fractions and calculate total protein in each. If purity of the Peak fraction >= 60%, go to step 10, if it is lower go to step 11.
Concentration. Concentrate protein from Peak fraction on a Viva Spin 20 with appropriate MWCO concentrator to 1.5-2 ml. Go to step 19.
Test Ammonium sulphate precipitation. Take a sample of the Sides fraction and add 0.8 ml of 4M ammonium sulphate to each 1ml of the sample. Spin down for 10 min at 19-24K rpm. Separate supernatant fraction and work out how much of the total protein has precipitated out. If you have more than 50% of total protein precipitated (presumably Target Protein is in the pellet) go to step 12, if less than 15% precipitated (you can presume Target Protein is in supernatant fraction) go to step 13, if more than 15% but less than 50% has precipitated, go to step 15. If you have used activity assay to monitor Target Protein, use it to reveal Target Protein distribution between fractions.
Test of Ammonium sulphate pellet solubility. Dissolve pellets in 1-2 ml of buffer A, spin down any insoluble pellets in the bench top centrifuge for 2min. Work out how much of the protein has been recovered from the Ammonium sulphate pellets. If there was poor recovery of protein from the pellet, go to step 16. If there wasn't very much insoluble material and recovery of protein from the ammonium sulphate pellet was reasonable, go to step 17.
Do not discard any of the pellets or supernatant fractions, keep them for gel analysis !

Hydrophobic chromatography

Test Hydrophobic chromatography. Wash 15-20ml Phenyl Toyopearl 650S column with 30-40 ml of buffer A and 1.8M ammonium sulphate. Apply Sides sample onto the column. Collect flow through fraction in the separate container. Elute with 150-200 ml of ammonium sulphate in buffer A (with a reverse gradient). Then wash column with 1CBV of buffer A and 3 CBV of water. Collect 6-8 ml fractions. Check protein concentration in every second fraction, including flow through fraction, buffer wash and water wash. There is a possibility that protein will bind to the matrix irreversibly. Run gel to analyse the elution profile. The gel should include: Crude Extract , 'Sides' before addition of Ammonium sulphate, flow through fraction (if there is any significant protein concentration), every second fraction containing protein, include buffer wash and water wash if there is any protein. Quickly stain-destain gel to reveal the Target Protein.
If there is no Target Protein revealed, it means that you probably can not use hydrophobic chromatography for this protein - go to Difficult Cases(2).
If you can find Target Protein on the gel, work out the Ammonium sulphate concentration required for its elution. Using refractometer and Ammonium sulphate-Sugar refractometer chart, measure Ammonium sulphate in the fractions with the Target Protein. Refine Ammonium sulphate gradient: it should start with Ammonium sulphate concentration being 0.2M higher than the concentration at elution point. If protein elutes early in the gradient (when Ammonium sulphate concentration higher than 1.2M) it is useful to get the final Ammonium sulphate concentration in the gradient to be 3 times lower than the starting one. If it elutes later than 1.2M, it is better to have buffer A without Ammonium sulphate as a final buffer. Go to step 14.
Main Hydrophobic chromatography. Take Peak fraction and add 4M ammonium sulphate solution to bring Ammonium sulphate in the sample equal to refined starting buffer. Save 0.1 ml of Peak fraction before addition of Ammonium sulphate for later gel analysis. To calculate volume of 4M Ammonium sulphate to be added to the sample (V_AS), use formula:

(V_S x SBC)/(4 - SBC)= V_AS,
Where V_S is volume of Peak fraction and SBC is Ammonium sulphate concentration (M) in the starting buffer.
Clarify sample by centrifugation for 10 min at 19-24K rpm. Apply sample on a Phenyl Toyopearl column and elute proteins with 200-250 ml of refined Ammonium sulphate gradient. Check protein concentration in every second fraction. If you find a distinct protein peak, combine 2-4 fractions with highest concentration and go to step 10 (concentrate protein on Viva Spin concentrator). If you are not sure, run gel to analyse each second fraction with protein. Also take for gel; Crude Extract , Peak fraction and MW markers. Combine 2-4 fractions with purest Target Protein and go to step 10. If protein was already purified by gel filtration, it should be pure - go to Finish. In the unlikely case that Target Protein is still not pure, go to Difficult Cases(1).
It is most likely that the Target Protein would start to precipitate at 1.8M Ammonium sulphate and would require a slightly higher Ammonium sulphate concentration for complete precipitation. Dissolve pellets in 1-2 ml of buffer A, mix with the supernatant fraction and add buffer A to bring Ammonium sulphate concentration to 1.5M, clarify by centrifugation and carry out test Hydrophobic chromatography, as in step 13, but the starting buffer should be 1.5M Ammonium sulphate in buffer A instead of 1.8M. Alternatively go to step 18.
In the rare event when Ammonium sulphate precipitation is irreversible, take 0.3 ml of the Peak fraction and add 0.1 ml of 4M Ammonium sulphate (this will form a 1M concentration of Ammonium sulphate). If protein precipitates go to Difficult Cases(3). Otherwise keep adding 4M Ammonium sulphate in 10-20 µl portions to find maximum Ammonium sulphate concentration at which the Target Protein still stays in solution. Consider this Ammonium sulphate concentration a starting buffer for test Hydrophobic chromatography. Take about 20% of the Peak fraction, add appropriate volume of 4M Ammonium sulphate to keep Target Protein in solution and run test Hydrophobic chromatography as in step 13 with appropriate corrections for the starting buffer. There is a high possibility that Target Protein will not bind to the column, if this is so go to Difficult Cases(3).
Ammonium sulphate precipitation. Take the Peak fraction and add about 0.85 ml of 4M ammonium sulphate solution for each 1 ml of the sample. Spin down pellets for 10 min at 19-24K rpm. Remove supernatant fraction and dissolve pellet in 1-2 ml of buffer A, spin down any insoluble material, check protein concentration, calculate total protein in the sample. You should have good recovery of protein in the sample, go to step 19, run SEC (gel filtration) and you will have your protein purified.

Ammonium sulphate cut

Ammonium sulphate cut. Perform analytical Ammonium sulphate cut (see Protocols) on a Peak fraction. If analytical experiment shows that it is possible to obtain more than 60% pure Target Protein using Ammonium sulphate cut, perform preparative Ammonium sulphate cut (see Protocols) then go to step 19 for SEC. If it is not possible to achieve 60% purity, go back to step 15.

SEC (Gel filtration)

SEC. Apply 1.5-2.0 ml sample of Target Protein onto a 1.6x60cm Hi-Load Superdex-200 column pre-equilibrated with buffer A and 0.1M NaCl. Run gel filtration at flow rate 1-1.5 ml/min. After 45 ml start to collect 2 ml fractions. Check UV elution profile. After UV peak is out of the column check protein concentration in each fraction across the peak. Collect no more than 25 fractions, as this is about 100ml, the elution point for a 5kDa protein. Run gel to analyse fractions containing the protein. On this gel there should be present: MW standards, Crude Extract sample, Sides sample, Peak sample, sample applied on the SEC column (if different from Peak) and each fraction across the protein peak (typically 6–10 fractions). When gel is completely destained, estimate purity of the Target Protein in the fractions. If you have fractions with the Target Protein of a suitable purity (>= 80% for crystallization if there are no major contaminations) combine 3-4 fractions with the most pure protein. Go to Finish. If protein is not pure enough, go to step 12.

Basic proteins

If Target Protein has pI higher or close to 7, the probability is that it does not bind to the DEAE-Sepharose column. However, some proteins despite having a high calculated pI have acidic domains and so they can be bound to the column. Collect flow through fraction and check protein concentration there by Bradford method. If you are monitoring Target Protein by activity, calculate total activity in the flow through fraction to be sure that all activity is there. If activity assay is not available rely on protein concentration in flow through fraction. If it is lower than 30% of the protein concentration in Crude Extract , it is likely that protein stays on the column. This is also true, obviously, if activity is not revealed in the flow through fraction. Go to step 6. If Target Protein does not bind to the column, go to step 21.
Tip: To find out quickly what part of the total protein does not bind to the column; take a sample (2-3 drops) of the flow trough fraction while Crude Extract is still loading and when UV absorption on the chart reaches a plateau. Check protein concentration and compare with Crude Extract .
Perform analytical Ammonium sulphate cut. (see Protocols). If precipitation of Target Protein is reversible, go to step 22. If it is irreversible, but Target Protein precipitates at Ammonium sulphate concentration higher than 1.5M, go to step 23. If Target Protein irreversibly precipitates with 1.5M Ammonium sulphate, go to step 24.
Perform preparative Ammonium sulphate cut (see Protocols). Dissolve Target Protein in 1.5-2 ml of buffer A. Relying on the analytical Ammonium sulphate cut you can make a good guess on how pure the Target Protein is. If purity is higher than 60%, go to step 19 for SEC. If it is lower, go to step 23.
Take about 25% of the Target Protein sample and run test Hydrophobic chromatography as in step 13, but the starting buffer should be 1.5M Ammonium sulphate in buffer A instead of 1.8M Ammonium sulphate. If Target Protein is revealed during test Hydrophobic chromatography go to step 14. If it is not revealed go to step 24.
It would seem that the Target Protein does not like Ammonium sulphate. Try cation exchange chromatography. Take 1-2 ml of flow through fraction and dialyse it against 100 ml of 20mM NaAcetate buffers each at pH 5.0 overnight. Clarify sample in refrigerated centrifuge, 15000-25000g for 10 min. Separate supernatant fraction from the pellet. Check volume of supernatant fraction and suspend pellet in the same volume of buffer A. Check protein concentration in supernatant fractions. Take a sample for gel analysis from supernatant fraction (10-20 µg of total protein). Take the same volume of the pellet suspension. Run gel and analyse distribution of Target Protein between pellet and supernatant fraction. In the best case, the Target Protein would be found in the supernatant fraction at pH 5 and many of the contaminants would be precipitated. Consider pH 5 for cation exchange chromatography (CEC) and go to step 25. If the Target Protein is a precipitate at pH 5 go to Difficult Cases(3).
Dialyse whole flow through fraction against 0.5-1litres of buffer B (starting buffer, 20mM NaAc pH 5.0), change to fresh buffer after 2-3 hours and leave overnight. Wash 10-15 ml column with SP-Toyopearl or S-Sepharose with buffer B. Check pH in the sample, and consider another 3-4 hours dialysis with fresh buffer if the right pH has not been reached. After finishing dialysis clarify protein sample by centrifugation (19-24K rpm for 15 min). Check volume and protein concentration and apply sample onto a column. Check protein concentration in flow through fraction to find out if the Target Protein binds to the column. If there isn't a significant amount of protein bound to the column, go to Difficult Cases(3). If there is significant binding, elute protein by 150-200ml gradient of NaCl from 0 to 1M in buffer B. Flow rate should be about 4 ml/min. Collect 5 ml fractions. Check protein concentration in fractions and analyse fractions with protein using SDS-PAGE. If Target Protein is pure enough - go to Finish, if it is not pure enough, go to step 26.
SEC. Concentrate protein on a Viva Spin concentrator to 1-2ml. Consider a buffer for gel filtration that is 1 pH unit lower or higher than pI and has 0.1-0.5M NaCl. Perform SEC as in step 19 but with appropriate buffer.
Cation exchange chromatography. As an alternative way for basic proteins you may consider to apply cation exchange chromatography as a first step. To allow protein to binds to cation exchange column you need to prepare crude extract at pH lower then 7. You can prepare crude extract directly in 50mM MES pH 6.5 or 6.0 or exchange buffer by dialysis. Than you proceed the same way, as for acidic proteins, but use SP-Toyopearl, CM-Toyopearl os S-Sepharose columns instead of DEAE-Sepharose column. Be aware that rather often basic proteins fail to bind to cation exchange columns with no apparent reason. However, if protein does bind to the column and you have it eluted with appropriate NaCl gradient (0-1M), the purity of the Target Protein could be high enough to consider SEC as a final step to obtain pure Target Protein even if initial expression level was not very high.

Difficult cases.

The Target Protein is not pure after 3 chromatography steps
Consider a fourth step.
If there is a HPLC system available, try Mono Q (Resource Q or any other anion exchange column) for acidic proteins and Mono S or analogies for basic proteins. Prepare sample in a low salt buffer. First perform a test run with 10-20% of the sample. For the test run consider a 30CBV gradient of NaCl from 0 to 1M in buffer A for acidic proteins and in buffer B for basic ones. Follow instructions that correspond to the column that you are using. Check protein concentration in peak fractions using chart. Run gel to see what level of purification was achieved. If you are not happy with the result, try a different pH (50mM MES-NaOH pH 6.5 is the first option). Optimise gradient. Run rest of the sample.
If there is no HPLC system, use low pressure equipment. Try anion exchange chromatography on a DEAE-Toyopearl 650S column or a Q-Sepharose column in buffer A. Also, you can change pH and run chromatography on either of the anion exchange columns at a lower pH. 50mM MES-NaOH pH 6.5 can be considered. For basic proteins try increasing the pH to 6-6.5. Do not discard flow through fraction as there is a high possibility that protein will not bind to the column. If protein is still not pure (and you haven't actually lost all of it by this time!) further options are HPLC Hydrophobic chromatography, chromatofocusing, preparative PAGE etc. In all my 25 year practice there has not been a single protein which has needed a fifth purification step.
Target Protein has not been revealed after Hydrophobic chromatography.
There are two ways to go: The first way is to do a second ion-exchange chromatography, as described above (See Difficult Cases(1)). The second way is to try a different type of Hydrophobic chromatography. Take 10-20% of the peak fraction for the tests.
The options are:
1. Use an Ethyl-Toyopearl column instead of Phenyl-Toyopearl;
2. Use 2M KCl in buffer A as a loading and starting buffer on a Phenyl-Toyopearl column or on a Butyl-Toyopearl column. The risk of losing the Target Protein on above columns is high, but if the protein is bound and eluted from them successfully, there is a high chance of achieving a good purification.
If Target Protein precipitates with less than 1.5M Ammonium sulphate or basic Target Protein precipitates with pH 5 or Target Protein does not bind to any column, this is, most probably, means that Target Protein is associated with small pieces of debris which are small enough to stay in the supernatant fraction during Crude Extract clarification. If there is a significant insoluble component of the Target Protein revealed in the solubility test this is another sign that, despite the appearance of Target Protein as a soluble protein in the Crude Extract , in reality the Target Protein was expressed as insoluble one. Try to optimise growth condition during Target Protein expression or try refolding from the inclusive bodies.

Finish

Target Protein

Appendix