Purifying a new protein is no easy feat. Finding combinations of protein purification buffer, salt, detergent, and stabilizing agent to get high yields of squeaky-clean protein can become tedious. Few things are as bothersome during this process as Heat Shock Protein (HSP) contamination. But worry not, we’ve got some handy tips to avoid HSP contamination in your protein prep.
What Are Heat Shock Proteins?
HSPs are protein chaperones that bind and stabilize polypeptide chains to help fold newly synthesized or denatured proteins. HSPs are found in all domains of life and their production rises in response to cellular stressors that cause protein unfolding and aggregation, such as heat. Unfortunately, HSPs can also spike when expressing recombinant proteins. And once HSPs are overexpressed they can be hard to remove.
Why Is Heat Shock Protein Contamination an Issue?
HSP contamination is a problem because it can interfere with downstream purposes. Structural studies require protein with >95% purity to ensure sample homogeneity. Lack of homogeneity prevents crystal growth in X-ray crystallography, complicates peak assignment in NMR, and distorts image processing in cryo-EM. HSPs bound to your protein can also block interactions that you’re trying to investigate using biophysical or biochemical assays. Thus, it is often worthwhile to optimize the removal of pesky HSPs.
HSP contamination can be stubborn because these proteins are very good at hugging substrates. Powered by ATP hydrolysis, they undergo a fast cycle of binding and unbinding short hydrophobic regions of substrate proteins. This problem is amplified when you’re trying to express a large protein and/or one that has unstructured regions. But no worries, there are many things you can do to pry your protein away from the clingy grasp of HSPs.
How Do I Know If I Have Heat Shock Protein Contamination?
HSPs are named after their molecular weight; so even a quick search for HSPs with a molecular weight that corresponds to a contaminating protein on an SDS-PAGE could be informative. Most HSP contamination will present as 40-110 kDa protein bands. The most common HSP contaminant when purifying from E. coli is DnaK, the bacterial homologue of human HSP70, which notoriously presents as a doublet around 70 kDa. GroEL, the bacterial homologue of human HSPs 60 and 20, is also a common contaminant and appears around 68 kDa.
There are various ways to confirm HSP contamination. Run a sample of your protein on an SDS-PAGE. Cut out bands of contaminating protein(s) and conduct mass spectrometry or N-terminal sequencing to quickly identify the contaminant. Alternatively, you could use a Western blot or an ELISA to confirm HSP contamination.
How Can I Decrease Heat Shock Protein Contamination During Purification?
Use ATP-MgCl2 washes
The most tried and true method for HSP removal is ATP-MgCl2 washes.1 Start your purification with an affinity chromatography step and supplement the washes with ATP and MgCl2. Because ATP stimulates substrate release, 20-column volume washes supplemented with 5-10 mM ATP and 20 mM MgCl2 can substantially decrease HSP contamination. ATP hydrolysis occurs rapidly, so perform fast washes (3-5 mL/min flow rate) or use a non-hydrolysable ATP analog. If you use an automated purification system and a pre-packed column, flow rate is controlled from the software. If you prefer to pack your own chromatography column, you can manually control the flow rate by adjusting the stopcock.
Adding a heat-denatured protein with a unique affinity tag to the ATP-MgCl2 washes can enhance results.  The idea is that you’re providing more misfolded bait for the HSP to pursue; that way it’ll let go of your protein and cling to something else. The denatured protein, and associated HSPs, can then be removed by affinity purification based on the tag. To remove GroEL contamination, supplement ATP-MgCl2 washes with purchasable GroES, which stimulates substrate release by GroEL.
Purify the Heat Shock Proteins
Flip the table and purify the HSPs instead! Many HSPs are purified using an ATP-agarose column. Pack a column with ATP-agarose resin and run semi-pure protein over the column. The HSPs should stick to the resin while your protein flows through. This is best done in between affinity chromatography and ion exchange chromatography. If you find that some of your protein sticks to the ATP-agarose column, elute the column with a salt gradient beginning from 0.05-1 M and pick the fractions that contain your protein. This may dilute your protein stock, but using ion exchange chromatography down stream will concentrate your protein.
Use an Elution Gradient
Experiment with an elution gradient. A shallow elution gradient across where your protein and HSPs elute can work wonders.
Use Zwitterionic Detergents
Add 0.001-0.01% of zwitterionic detergents, particularly during cell lysis and the first affinity purification step. Zwitterionic detergents have both positive and negatively charged atomic groups with an overall neutral net charge. The dual charge allows zwitterionic detergents to efficiently break apart protein-protein interactions similar to ionic detergents, but the neutral overall charge prevents modifying protein structure or charge, as would an ionic detergent.
Optimize Construct Design to Limit Heat Shock Protein Contamination
- Design a new construct of your protein. Most times, simply using a protein construct that omits unstructured, hydrophobic regions that attract HSPs will fix the issue. To identify these regions, look at sequence conservation, secondary structure prediction, and homology modeling. Keep in mind that this step may not always be feasible. If the unstructured and/or hydrophobic regions are necessary for functions that you’re investigating, such as protein-protein interactions or catalytic sites, construct re-design is not a good option. But if you’re determining a structure or function that does not require these regions and is not affected by their removal, construct re-design is an easy and effective alternative.
- Bacteria have trouble correctly folding recombinant proteins with complex structures. In this case, HSP production will increase to fold your protein and/or prevent its aggregation. To remedy this, express your recombinant protein with an N-terminal fusion protein that is easy to fold in order to provide momentum for correct folding. The maltose-binding protein (MBP) and Small Ubiquitin-like Modifier (SUMO) tags are excellent for improving protein expression, solubility, and folding of structurally complicated protein.
Modify Protein Expression Conditions to Reduce Heat Shock Protein Production
- Optimize time and temperature of induction to maximize recombinant protein expression and minimize HSP production. Sometimes, induction at low temperatures (16-18oC) for a longer amount of time (17-20 hours) is best because it slows down the accumulation of recombinant protein. Alternatively, induction at higher temperatures (30-37oC) for short timeframes (3-6 hours) can generate large quantities of your recombinant protein before HSP production spikes.
- Try a different cell line or switch expression systems. Common bacterial expression strains with HSP knockouts exist, such as a BL21(DE3) strain with a DnaK knockout. If bacterial expression is troublesome, try moving to human cells. Thermo Fisher Scientific’s Expi293T cell line is a robust, suspension-based expression system. If you do not require high yields of protein, in vitro translation is a great option to generate your protein.
Although HSPs are clingy, there are ways to eradicate or at least substantially decrease their grasp on your protein. With a little patience and a couple of the above-mentioned suggestions, your protein will have a much better before and after photo.
- Rial, D.V., and Cecceralli, E.A. (2002) Removal of DnaK contamination during fusion protein purifications.Protein Expr Purif. . 25:503-7.
- Ratelade, J. et al. (2009) Production of Recombinant Protein in the Ion-Deficient BL21(DE3) Strain of Escherichia coli in the Absence of the DnaK chaperone. Appl Environ Microbiol. 75:3803-7