Title: Protein Purification

Techniques to Master:

Bacterial lysis, membrane and nucleic acid removal, affinity chromatography

Learning Objectives:

1. Describe how to choose a purification procedure

2. Explain how to prepare bacterial cell lysates

3. Describe how to plan purification by affinity chromatography and how to perform it

Background:  

Gravity-run liquid chromatography for protein purification relies on the passage of a sample of mixed proteins over a bed of highly engineered particular agarose resin that has been carefully poured into a glass or plastic cylinder.

For gel filtration, proteins are separated based on their size. Small proteins travel through minute spaces in the agarose beads while the larger proteins are excluded. For this type of column, all the proteins pass through eventually, without the addition of a compound that competes with the protein for binding to the resin.

Ion exchange chromatography and affinity chromatography depend on an agarose resin having a ligand that binds the protein of interest. The protein is later eluted from the resin by application of a compound that competes with the ligand. In ion-exchange chromatography the eluting compound is often a salt but can be a buffer with a different pH that changes the charge state of the bound protein. In affinity chromatography the eluting compound can also be a salt but is very often a compound that resembles the ligand covalently attached to the resin. For example, proteins that bind nickel or cobalt-containing resins via their his6 tags are often eluted with imidazole. Our curriculum, as its goal is to purify a wide variety of proteins, will encounter different methods of purification. This handout aims to introduce you to the methods you will use to decide what will work best to purify your unknown protein of interest. You will have to consult resources outside of this document since your project is so original.

Before you begin to plan, consult a plasmid map of the plasmid containing the coding sequence for your protein of interest. pET plasmids were first patented by Novagen Corporation; now, maps can be accessed on the EMD-Millipore website as well as others. pET vectors all depend on transcriptional control via the T7 promoter, but only some contain tags. Some of them code for his6 tags and others for protein fusion tags. Protein fusion tags are coding sequences for other proteins that may facilitate purification or stability. An example is the maltose binding protein (MBP), which enables purification utilizing MBP ligands, and also can enhance stability of the protein of interest. You need to know if your protein is going to be expressed with such a tag. Furthermore, many new vectors are based on pET vectors and have not just protein fusion tags but also protease sites that will allow removal of the fusion protein in case it hampers the native structure or function of the overexpressed protein. Some examples are pMCSG vectors, which are described here: http://bioinformatics.anl.gov/mcsg/technologies/vectors.html

 

A. Choosing a method.

Figure 1. Metal (in this figure, nickel) affinity resin structure. The circle represents the agarose resin bead. The nickel or cobalt immobilized on the resin co-ordinates to two histidine residues of the his6tag. (The gray line represents the peptide backbone of the his6tag.)

B.  How to make figures and tables for protein purification.

Several data sets and representations are standard in protein purification. One is a purification profile. This document displays the total protein content, the particular protein content, and the specific activity, at every step of the purification procedure [1]. The specific activity (quantity of enzyme of interest divided by quantity of total protein) is a measure of how enriched a sample is in the target enzyme. As contaminant proteins are removed, this ratio goes up, until it reaches a value that does not change because the enzyme has been purified to homogeneity. For the purpose of keeping this data set complete, small samples of protein must be saved from every step for later enzyme assay and protein assay.

Another usual feature of a purification description is some representation about the progress of a chromatography step. Similar to the overall purification profile, it keeps track of the levels of enzyme of interest and of total protein in every sample that results from the chromatography exercise. It includes the starting material, the sample of materials that did not bind, and the fractions collected upon elution of the resin.

Purpose:

When a biochemist prepares to study an enzyme, she/he purifies it as soon as possible. In decades past, the reason was partly to get its physical characteristics, such as pI or molecular weight. In our curriculum, the enzyme has been cloned and sequenced, so these parameters are already available. However, a biochemist still requires pure materials for study, with the rationale that contaminants (protein binding partners, inhibitors, substrates) must be removed as variables that cannot be controlled. This is especially true if your goal is to find out what the function of the enzyme is. It must be separated from all of the E. coli enzymes. Therefore, the first thing to do is purify the overexpressed protein. Other compounds in the bacterium may alter any assays or studies done before the protein is pure at the time of harvest.

Experimental Design Considerations:

As alluded to above, considerations include [2]:

Supplies:

Equipment:

Sonicator for lysis

Centrifuge for membrane removal

Chromatography columns (and fraction collectors, depending on scale)

Spectrophotometer for quantifying protein and activity

Reagents:

Buffer (HEPES is often a first choice due to stability of its pKa. The pKa of Tris changes with temperature. Phosphate may be an inhibitor, activator, or reaction product.)

Additives and stabilizers (chosen as described above)

RNAse, DNAse, (benzonase®, EMD BioSciences) or streptomycin sulfate

Chromatography resin (kits will be suggested in this document but all are                 available for bulk purchase)

Elution buffer

The contents of this buffer will depend on the resin utilized. It might be simply salt, or might be maltose (for MBP) or imidazole (for histidine6)

Procedure:

A. General Steps:

1. Lysis

to use.

2. Centrifugation

3. Nucleic acid removal (optional—if the lysate is very viscous. Beware that precipitating reagents might have to be removed for binding to chromatography resin in the next step. See PD-10 column use in “supplement”)

        

4. Chromatography

This step will be very different depending on what scale is chosen. Small-scale chromatography can be carried out in 1.5 ml microfuge tubes or 1-ml syringes pre-packed with small glass beads, then chromatography resin, and larger-scaled runs can be carried out in the five-to-ten ml columns marketed by several biotech firms. It will also be very different depending on what protein or affinity tags are present on the cloned enzymes of interest. Possible resins and vendor sources are listed at the end of this document in “Supplement”. Whichever size or resin is chosen, the following steps are the same for every liquid chromatography process used to purify proteins.

B. Trouble-shooting

1.        What if your protein did not bind?

        Consider the ingredients in the starting sample, i. e., the lysate that you applied to the column. Might something have interfered with binding?

        Consider the capacity of the resin. Maybe your protein did bind but there wasn’t enough resin for all of it.

2.         What if your protein did not elute?

        Consider the ingredients in the eluting buffer. Maybe the reagent that competes with the resin’s ligand was not concentrated enough.

3.        What if your protein elutes but it is not pure?

        Consider whether buffer flow through the column was interrupted by cracks or resin shrinkage.

        Consider whether another step must be added. Sometimes another chromatography step must be performed, or, the same resin used again but with a different elution strategy, such as a pH change instead of a wash with competing ligand.

4.         What if your protein elutes but it is not active?

        Consider adding stabilizers such as were mentioned above to your buffers.

        Consider doing everything in the cold as much as possible.

        Consider using a protease to remove the protein tag that allowed purification. TEV, TVMV, or enterokinase protease cut sites are often engineered into plasmids to make this possible.

Clean-up:

Centrifugation steps result in pellets that must be treated as bacteriological waste, since they can contain unbroken, viable cells. Centrifugation plasticware must be cleaned as one would clean biological waste, with bleach or strong detergent and hot water.

Samples should be saved and frozen in the coldest freezer available until they have been analyzed for content.

Most affinity resins are agarose beads with ligands attached. As such they are an excellent energy source for fungi and bacteria, and will grow these organisms unless stored in a clean state. They can often be recycled and used again for significant economic savings. Follow product instructions for recycling and storing the resins.

Interpreting Results: 

Scheduling challenges may prohibit the collection of data during the same class period as the chromatography is performed. Still, it will usually be possible to detect the presence of protein in certain samples (e. g., the eluates) quickly by reading the A280. Eventually specific activities should be determined for every sample, as described above, so that the purest samples can be saved for further analysis. Samples will all be exposed to SDS gel electrophoresis as well, in corroboration of the purities obtained by specific activity.

References:

  1. Burgess, R. R. (2009) Chapter 4 Preparing a Purification Summary Table. in Methods in Enzymology, pp. 29–34, Elsevier, 463, 29–34
  2. Linn, S. (2009) Chapter 2 Strategies and Considerations for Protein Purifications. in Methods in Enzymology (Deutscher, R. R. B. and M. P. ed), pp. 9–19, Guide to Protein Purification, 2nd Edition, Academic Press, 463, 9–19

Creative Commons LicenseThis work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

Supplement:

If a sample should be exchanged into an appropriate binding buffer (for example, for salt or streptomycin removal)  before application to an affinity column, this can be quickly accomplished using PD10 columns from GE Healthcare. Description of these columns and instructions for use can be found at:

http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-us/products/AlternativeProductStructure_17402/17085101

The likely constructs encountered in this curriculum are listed here with sources of affinity resin:

For a his6-tagged protein, options include Talon resin, with resin or kits marketed by

1        Clontech:

http://www.clontech.com/US/Products/Protein_Expression_and_Purification/His-Tagged_Protein_Purification/Cobalt_Resin-Batch

2        Or Ni-NTA resin from QIAGEN:

https://www.qiagen.com/us/shop/sample-technologies/protein/protein-preparation/ni-nta-agarose#orderinginformation

3        or Prep-Ease resin from Affymetrix:

http://www.affymetrix.com/estore/browse/brand/usb/product.jsp?navMode=34000&productId=131373#1_1

Instructions accompany the materials purchased.

For a MBP-tagged protein, options include

4        NEB, amylose resin:

https://www.neb.com/products/e8021-amylose-resin

5        Dextrin-Sepharose from GE Healthcare:

http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-us/products/AlternativeProductStructure_17340/

For Glutathione-S-transferase tagged proteins

6        Glutathione-sepharose from GE Healthcare:

http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-us/products/AlternativeProductStructure_17325/

For Calmodulin-Binding-peptide (CBP)-tagged proteins:

7        Calmodulin resin from G Biosciences:

http://www.gbiosciences.com/ResearchProducts/Protein-Research/Purification-and-Chromatography/Affinity-Purification-Resins/CBP-Tagged-Protein-Purification.aspx