Paul.xu at 18:37, 9 May 2013

2013-05-09T18:37:08Z

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== Expression ==

'''Media''' – Don’t settle only for LB and IPTG induction; you should try the Autoinduction media, developed by William Studier, which can boost protein expression between 3-10 fold, depending on the protein.

'''Temperatures & induction time''' – Check several temperature points: 18, 27, 30 and 37 degrees Celsius, while varying the induction time appropriately. Meaning, the lower the temperature the longer induction time (we routinely induce proteins at 20 or 27 degrees over weekend). Start with an overnight and modify accordingly.

'''Strains''' – Try inducing with Rosetta, Codon+, Tuner and Rosetta-Gami strains.

== Protein Purification 3 Steps ==

=== Capture ===

 Affinity chromatography (AC), Ni Ion exchange chromatography (IEX), SP FF.

 Centrifugation at 45,000 rpm for 30-45 minutes is sufficient for most proteins (you can live also with half of it using SS-34 rotor at 20,000 rpm for 2 hours).

If using a dripping column, initially pack beads that will be sufficient to ~10mg protein/1L of induction. After the first purification it will be easier for you to adjust the amount of beads to get the best purification without getting too much junk.

Remember that in most cases the affinity chromatography step will determine the fate of your sample in terms of purity levels. Do your best to calibrate this step to the optimal setting possible so you get the protein at the highest purity level. From my own experience a dirty sample post affinity will not improve tremendously after using other columns (again, in most cases).

Perform extensive washes, especially if working with dripping columns, 20 times the amount of beads volume (20 CV), varying the amount of salts and Imidazole.

Elution should be conducted at a relative small flow rate to get the sample concentrated at the end of this step; in certain cases you’d want to increase the flow rate to avoid high concentration elution in case the protein is prone for aggregation.

Evaluate your sample purity through an SDS-PAGE analysis. In case you find it hard to identify your protein do a western blot for verification (blot directly from the coomassie-stained gel).

Depending on the purity level required and the observed purity of the sample, assess whether you want to do a step of ion-exchange or size exclusion.

In case you plan on doing a step of ion-exchange, you might consider eluting your protein with low salt concentration (maximum 50 mM ion strength) and continue directly with your post-affinity elution into the ion-exchange column.

 

Nickel affinity column gradient elution – capturing and purifying

While batch binding and elution step can be performed on a faster time scale (hour to hour and half from start to finish including wash steps), using a prepack nickel affinity column (such as HisTrap from GE) can help ending your first chromatography with a higher level of purity. This is true for all samples but it is crucial when you find your nickel affinity purification relatively dirty.

Why not use the prepack column from the first place?

It depends on the amount of proteins your purifying at a single moment (in our lab we sometime purify two to four proteins in parallel) and of course, if your sample volume is large it will take more time to bind, wash and elute.

When I am using a prepack column with gradient I still get the protein dirty no matter how much wash steps I do – what can be done to improve the nickel affinity purification?

From my own experience, when you have a good overexpression (i.e. higher than 15mg/1L of induction) then you will have better purification because the dominant protein in the sample will be your protein. However, if you have a low expression from the first place then your protein will have to compete with other host proteins which have similar expression levels. In such cases you should first consider improving expression via Autoinduction media (Studier et al. 2005) which I found to increase the fold of protein expression by 3-10 fold, depending on the protein. See my previous note about increasing expression.

Assuming you’ve tried everything and your protein is still low expressing you can do the following combination strategies, separately or combined:

Decrease beads volume – if using a prepack column, try loading the protein on a 1ml column and not a 5ml. Why do that? Well, let’s assume your protein is expressing at 5mg/1L of media and you induced 4L of culture, giving a total of 20mg of protein of interest. If you will load the lysate on a 1ml of prepack column, that can capture 20-30mg of tagged protein, between 67% to 90% of the column will be captured by your protein. On the other hand, if you load the 5ml prepack column, with a potential capacity of 200mg (!), all the rest of the host proteins will have plenty of room to bind to.

 Removal of periplasm – Another strategy is to do periplasm wash of the bacterial pellet before performing the bacterial cell lysis. That’s because the periplasm was shown to contain many proteins and enzymes that naturally bind divalent/metal ion. By removing these protein you lower the host protein’s that compete over binding to the beads.

*Spin down cells, then suspend cell pellet with 100 mM Tris-HCl or Tris-Acetate (pH 8.0) containing 20% sucrose and 0.5 mM EDTA and incubated on ice for 15 min.
*Spin down cells again, the supernatant (sucrose wash) contains periplasm proteins.
*Resuspend cell pellet again in ice-cold deionized water and incubated on ice for 15 min.
*Spin 6,000 rpm for 5 min, the supernatant is combined with supernatant from the sucrose wash---the combined fraction has most of periplasm proteins.

 Guanidinium/Urea wash – This is a bit risky but it is worth the try if protein is for structural studies. With this strategy cells are lysed and bound to the nickel column. Then a wash with 2M of Guanidinium or Urea is performed which usually removes most of the host proteins and afterwards a shallow gradient of decreasing amounts of Guanidinium/Urea brings back your protein to native state. Of course, you will need to check and see that your protein didn’t loss its fold and function (usually if the protein refolds improperly you will notice aggregates once you concentrate your protein). 

=== Intermediate Purification ===

Hydrophobic interaction chromatography (HIC), Phenol FF, MEP Ion exchange chromatography (IEX)

After doing our best to purify our protein at the affinity chromatography step, we might decide we want an additional step of purification, which in many cases involves either an Ion-exchange or size exclusion chromatography. In this part I will focus on Ion-exchange chromatography and some tips on how to maximize your time and effort to get the most of this system.

 Unlike affinity chromatography, the elution in IEC is conducted via increase in ion strength or gradual change in pH that may affect your protein behavior (structure and/or function). Since binding of the sample to the column should be at minimum salt interference and maximum surface charge, usually there will be a requirement to lower the salt concentration. If the previous step was a His tag Affinity chromatography, it will save time if the elution step will contain a minimal amount of salt (e.g., ~50mM NaCl) so there will be no need to exchange buffer. If you’re wondering about the effect of the Imidazole in the solution, it depends on the type of IEC column used. If you’re using a cationic exchanger or working under pH 7 you have no need to exchange the imidazole simply because the fact that imidazole has a pKa of ~7 for its first nitrogen, thus at higher pH the ring will not be charged and will not interfere with the binding of the protein to the matrix.

The beauty about IEC is the fact it is a capture method: you can load as much volume as you want as long as you don’t overload the column with protein mass. This means you can start from 50ml or even 500ml diluted solution assuming you load your sample though a buffer valve. One thing to remember, though, is that there are proteins which do not bind to the anionic or cationic (or both!) so make sure you start collection immediately upon injection. Even in case your protein was not bound to the column, the contaminants might bind to the column so it is important to step-elute the bound material and evaluate it against the flowthrough and the pre-injected sample.

 

'''IEC elution step'''

When considering the elution step, two primary factors should be evaluated: elution buffer composition and elution methodology. In regard to the elution buffer it is usually recommended to use high concentration of salt (1-2M). pH shifts is less common since proteins are sensitive to small changes to the buffer’s pH which might affect their structure. On the other hand, it should be considered that gradual increase in salt can shift the protein from one oligomeric form to another (i.e. monomer vs. dimer) depending on its inter-molecular surface characteristics. Exploring other salts than the common NaCl is recommended when the protein aggregates or not eluting from the column.

The most common elution methodology is gradual increase in eluting buffer concentration (commonly known as “B buffer” or %B). While requiring more time, this methodology is essentially “fail-proof” as long as the gradient is relatively shallow such as 50%/50 min or 50%/10 column volume (CV) at 1ml/min flow rate using a 5ml column. Essentially you’ll want to elute the protein across 10-20CV so you’ll have better separation among close peaks. The slow rate will generate a much more concentrated elution which can expedite downstream concentration step.

Usually it is recommended to do a pilot run on the IEC. Inject small amount of the sample so you won’t overload the column (depending on the amount at hand, try to aim at 5-10mg) and evaluate the separation profile established with the current elution buffer composition and gradient. It is essential to couple a SDS-PAGE analysis of selected peaks together with the chromatogram so you can evaluate the following parameters:

 

'''Peak separation'''

 Purity of the selected fractions Amount of injected sample – should you increase/decrease the amount?

At this point you can decide on keeping or changing the current settings.

When analyzing the peaks, you should remember that proteins can bind to the matrix through differently charged surfaces, which means that the elution profile of a 95% protein can generate several elution peaks. That’s why it is crucial to perform an SDS-PAGE analysis of the major peaks to verify whether a certain elution contains the protein of interest or a contamination. You should also remember that in certain cases what looks like a monomer on the gel can well be a dimer or an oligomer which forms a new charged patch that doesn’t exist in the monomer state; in certain cases, like crystallography, sample heterogeneity at the oligomeric level can be fatal for successful crystallization effort.

'''Step or gradient?'''

Moving from the gradient methodology to the step elution is a bit more complicated than it seems, especially when trying to separate between closely related peaks. Lets take the following scenario on the right.

[[Image:ProteinPurification_example1.jpg]]

In this purification run the main peak is bordered by two relatively small peaks. This was done by using an anion exchange column; color codes are blue color stands for 280nm, red for 254mn and brown for the conductivity in mS. Even though I would not recommend a shift from gradient to step in this particular case, I will use it to explain the workflow to perform such a change. First you should determine the volume in which you will want to start the step elution according to the absorbance curve (in this case I chose the inflection point, red dash line). From this volume deduct the total volume of the column (in this case 5ml) + additional 10% overhead in case of measurement errors. An additional way to determine the exact volume is to use the “%concentration” curve and determine the difference in volume between the starting point of mixing buffer A and B and the point at which there is increase in conductivity. I have done so manually (without the %concentration function) and established the volume at ~115ml (blue dashed line) afterwhich I have deduced the conductivity value at this specific volume. This will be the first elution step that will remove the preceding contaminant peak. The same can be done to separate the main peak from the late contaminant peak. Once you figure this out, repeat the test run with this step elution and see if it fits with your needs.

=== Polishing ===

 Gel filtration (GF)

HiLoad 16/60 & 26/60 Settings that improve purification by Size exclusion chromatography

Having the best column for your needs doesn’t necessarily mean that your purification step will be ideal with ~100% resolution (the measure of separating one specie from another). In many cases some contaminates will be evident, some even in a cryptic shoulder that will be very difficult to observe. Here are the main settings you should give attention before running a separation:

• Load – avoid overloading the column with your precious protein unless you know there will be no close species to your protein’s elution volume. Seek advise with the column’s data sheet in regard to acceptable injection volume and protein mass. Measure the sample’s concentration and if the sample is not pure add 10-20% mass to your final protein concentration (according to the amount of contamination. As a rule of thumb your protein sample volume and mass for injection should be in the range of 0.5-3% of the column volume and no more than one tenth of column volume in terms of mass. This means that if you’re using a 24ml column, use 120-500 microliters of protein sample with a maximum protein mass of 2.4mg. Again, these estimates are for your first run and if you find you can inject more protein mass, then go ahead. However, if you find a cryptic shoulder (a sign of a closely eluting specie) then you might want to lower the amount of protein you inject. It will be easier for you to cut out a small peak of several injections than trail than a larger trail in a single overloaded injection.

• Flow rate – chromatography is based on the dynamic transition between equilibrium and disequilibrium between the mobile and the stationary phases (“theoretical plates”). For this equilibrium to occur, sufficient time should be supplied. With respect to our modern FPLC systems, this means a reasonable flow rate, which depends on the specific column (usually no lower than 0.2ml/min or higher than 4ml/min).

• Mid-run injection – In cases you have to do many injections you might want to explore mid-run injection. As the name implies, after performing the first separation analyze whether you can inject an additional sample before or after the expected elution volume. For example, if you have an elution profile of peak A eluting at 7ml with peak width of 1ml, peak B starting eluting at 12ml with a peak width of 1ml and peak C eluting at 20ml with peak width of 0.5ml, you can safely inject two injections 2ml apart. This technique can save a lot of time when the chromatogram is not crowded with many peaks.

• Consecutives SEC columns – In case you have a combination of complexes (80kDa) and monomers (40kDa) you might want to explore the use of two complementary columns, such as Superdex 75 and Superdex 200. Assuming you’re aiming at isolating the complex from the monomers and the other small contaminants, the first separation should be performed with Superdex 75, collecting the complexes eluting at void volume and then reinject the elution into Supderdex 200 to separate them from the other contaminates. This technique is especially effective when it is hard to separate complexes from monomers in a single column.

Convert linear flow units (cm/hr) to volumetric flow units(ml/min). Formula for the conversion from cm/hr to ml/min (VF): VF=0.013*X*d^2 where X is the linear flow value and d is the internal diameter of the column in centimeters. 

[[Category:Knowledgebase]]

Protein Purification 3 Steps - Revision history

Paul.xu at 18:37, 9 May 2013