Bench Tip: Tips for Performing Preparative Chromatography and Protein Purification

Abstract

Separating out a protein from a complex mixture is not an easy task. Many hours are spent by bioprocessing scientists trying to purify their favorite protein to homogeneity in order to study the functional properties or to manufacture a large-scale preparation for production or potentially clinical use. Even 1% contaminant in an otherwise highly purified protein preparation, can reek havoc in a functional assay or structural protein study not to mention in the drug discovery process. 

As many researchers have learned, the purification process is as much of an art as it is science. As with the more common artistic endeavors, protein purification not only relies on protocols and procedures, it brings together intuition, years of practice and trial and error experimentation. While there are different procedures to purify protein, in today’s lab, the method of choice for separating and purifying a target protein of interest from complex mixtures is liquid chromatography. Here we provide some fundamental tips and tricks to preparative chromatography that will yield a pure sample preparation that will advance your protein characterization studies.

Know what you are after

The fundamental strategy of protein purification is simple— remove all contaminants to enrich for the amounts of your protein of interest before transitioning to a large-scale preparative purification. Once you understand the physical properties of your target protein, you can scale up the process using preparative chromatography. Know the relevant properties of your protein such as pI, molecular weight, and hydrophobicity. This will assist you in developing a purification strategy.

Tip 1: When creating methods and protocols, try to minimize redundant steps. This will provide clarity and accuracy to the protocol and reduce operational mistakes. Creating method templates is also a great way to save setup time and eliminates the need to monitor the entire run.

Assay method

Once you understand the physical properties of the compound you are trying to purify, you should select one or two assay methods for detecting your protein during the purification steps. Before embarking on any protein purification project, it is a good idea to select an appropriate biochemical assay to detect and quantify the target of interest. This helps during method development in obtaining the right compound and it helps to monitor purity during each step. For example, if you are purifying an enzyme, an enzyme specific biochemical assay could be used to monitor activity. If the molecular weight of your target is know, you could use either staining or aWestern blot as confirmation. Or, if you understand the spectral properties of your protein, absorbance with a spectrophotometer would be used

Lysate clearing

Let us consider the facts, rarely are sample preparations simple and homogenous, typically they are very complex hetergenous mixtures. Having too much debris in a lysate that is loaded onto a column is a great way to clog your system and ruin your run. One method that can be applied is to treat the lysate with Benzonase® endonuclease  or DNase I to chew up the nucleic acids and reduce the viscosity of the sample. Keep in mind that you have introduced an enzyme into your sample, so follow the manufacturer’s suggested use protocol.

Another popular procedure to clarify lysates prior to the initial capture step is to centrifuge at 45,000 rpm, for 30 minutes with a 45 Ti rotor or equivalent. In addition, filtering samples through a 0.45 or 0.2 μm filter is sufficient to remove particulates. If your sample does not flow easily through one of these filters, try a 0.8 μm filter. The larger size works well for proteins with visible particulates and those proteins that don’t agree with the smaller pore sizes.

Tip 2: Be sure to select a low protein binding membrane such as PVDF (polyvinylidene fluoride) or PES (polyethersulfone) as nylon filters often bind protein causing a loss of yield.

Protein Storage

Generally, proteins are best stored at ≤ 4°C in clean, sterilized containers or tubes. Storage at room temperature often leads to protein degradation and/or inactivity. For short term storage of one day to a couple weeks, store the preparation in simple buffers containing protease inhibitors at 4°C. For mid-term storage-1 month to a year, freezing at -20°C or -80°C is recommended. For serious long-term storage of up to one year, some researchers choose to bead single-use aliquots of the protein in liquid nitrogen. Depending on your situation and the properties of your protein, you might favor one storage temperature and storage duration over another.

Tip 3: Be sure to keep in mind the downstream applications as well as some proteins will not respond well to freeze/thaw events.

In addition, many different compounds may be added to protein solutions to extend storage life. What you add to your protein solution depends on the intended use and the characteristics of your purified protein. There are many additive choices and vendors such as Bio-Rad offer reagents in convenient sizes and have protocols and tips for which additives to use. Be sure to read and plan your downstream experiments to ensure the proper storage conditions.

Column and system tips

First and foremost, be sure to maintain your column and system as a whole. Columns and systems are expensive, so like with any other investment, you should take good care of those you have. By following the simple tips below, you can be sure to extend the life of your column and system.

Use of an inline pre-column filter or guard (20 μm) between your inject valve and column is an effective way to increase column lifetime. Also, check for worn frits and O-rings in manually packed columns and replace them when you find them. This will prevent possible pressure problems. Likewise correct storage of your column will help high pressure instances due to excessive bacterial growth build-up in the column. Bacteriostatic solutions should be run through the column to prevent potential contaminants. This step should be followed by running water through the column as a final flush to remove any aggregates that form at the top of the column.

Other places on your system that need care are inlet lines, loops, and tubing. Periodical replacement of tubing that comes in regular contact with sample, especially lines connected to your fraction collector. The tubing surface is exposed to large volumes of protein-rich solutions that can start to clump and eventually clog the line. Lastly, periodically cleaning sample inlet lines and loops with 1N NaOH solution followed by water or neutralizing buffer will also help eliminate sample buildup in the tubing. For best results, occasionally sanitize your entire system with 1 N NaOH to clear the flow path from pump to fraction collector and store all system lines in fresh 20% ethanol when not in use to avoid bacterial growth.

(Note: if your system contains a pH probe, we recommend taking the probe out of line before performing the treatment

Tip 4: Periodic checks for cracks and bends in system tubing will help avoid restricted flow leading to increased system backpressure.

One final note, before staring your initial run, check your system pressure when first running a column and record it in a place that you can easily find. You can use this comparison in the event you notice something wrong with your run.

Odds and Ends

There are many ways to maintain your system so that your runs are consistent and reproducible and we have touched on a few main points above. These additional notes, will get you going on your journey with preparative chromatography.

  • Column switching valves save setup time when multiple projects are performed on the same chromatography system
  • Loading lysate through sample pumps is preferable over using system pumps. The latter increases the chances of sample buildup and contamination in your system
  • Buffer blending is an effective way to reduce both solution prep and method development time
  • To achieve optimal gradient performance, choose the mixer size that corresponds with the operating flow rate.
  • Proteins and nucleic acids can be monitored together with a multi-wavelength detector. You can also analyze their relative proportions using software for automated chromatography systems.

Whatever your reason for doing preparative chromatography, the one thing that will keep your experiments going is taking care of your system. Preventative maintenance is the best way to keep your experiments on track. Consider using the websites of vendors such as Bio-Rad to discover tips and guidance in selecting the latest equipment and reagents that will make protein purification easy, and provide insightful tips on setting up and running your instrument. They may even be able to provide custom solutions tailored to your target protein of interest.