The Biochemistry Behind Protein Purification

Cell-free protein expression is a versatile tool for producing proteins at the location where they are needed, in a purity that is not reached with conventional methods. But as this technique is susceptible to changes in the experimental conditions they have to be optimized thoroughly. So we decided to use conventional expression in Escherichia coli to reliably get higher yields of protein for testing and optimizing additional to cell-free protein expression.

Using the Natural Expression Machinery: Growing Cells

Figure 1: Schematic overview of protein overexpression.Transformed E. coli are grown to an OD of 0.5 and then induced with IPTG. After a certain time the cells are harvested and can be stored frozen.

The genetic information for our antigens was transformed into the bacteria we used (see: strains we used). As production of foreign proteins (heterologous expression) can cause severe stress in the cells, the expression level is controlled by using the lac-repression system. Only after addition of the non-degradable allolactose analogon IPTG (Isopropyl- thiogalactopyranosid) the lac-repressor dissociates from its binding site and gives way for transcription. For fast expression the viral T7 RNA polymerase is used instead of endogenous polymerases.
To yield optimal protein concentrations each expression system first has to be optimized in terms of temperature and IPTG-concentration as both factors strongly influence yield and solubility of the produced proteins. This is done by starting with small scales and testing a series of conditions while the yield is quantified by SDS-PAGE. Once the optimal condition is found, the scale is increased and the proteins are purified to a purity that is suitable for our experiments.
In our basic work-flow cells are first grown to an optical density of 0.5 before they are induced by IPTG (figure 1). This is done to ascertain that there are enough cells to overcome the stress caused by induction. After a certain expression time (two, four or eight hours) cells are harvested by centrifugation and can be stored by freezing ¹⁾²⁾.

Getting Protein Out of Cells

When cells are destroyed and proteins are released into solution they are instantly degraded by proteases. These enzymes do not get in contact with the target protein in the intact cell. But in the disrupted cell the degradation has to be prevented by addition of inhibitors. We used PMSF, a small organic molecule that specifically inhibits serine proteases, by adding it to the solution just prior to cell lysis ³⁾. Cell lysis was performed by sonification, the use of ultrasonic pressure to mechanically destroy the bacterial cell wall. To remove most of the cell debris, the crude lysate was first centrifuged, the supernatant was transferred into a new tube and a second centrifugation was applied.
To pellet organelles and membranes, higher centrifugal forces had to be applied. The target proteins can be found divided between the pellet (inclusion bodies) and the supernatant (soluble fraction). The soluble fraction contains natively folded proteins and was used for further purification.

Create a Useable Protein Solution

Figure 2: Comparison of the structure of Imidazole and Histidine.

Even after centrifugation the protein remains far from pure. To specifically enrich the protein of interest, many different affinity-based methods have been developed. Therefore, a decahistidine tag is fused to the protein. The interaction of these amino-residues with divalent cations like nickel allows for selective binding to a stationary phase. The protein solution then is pipetted onto an agarose matrix with nickel ions coordinated to NTA-groups. Several washing steps remove unspecific binding of other proteins and the antigen is finally eluted with imidazole that competes for the binding to the cation (figure 2). After elution the protein is more pure, but also the concentration of imidazole, used for elution, is strongly increased (figure 3).

For our project, the expressed proteins are supposed to be immobilized on a glass slide. The expressed proteins are fused to a His-tag, whilst the glass surface bears the respective catchers for the tags like a Ni-NTA-modified surface. Therefore, we are able to specifically bind the antigens onto the slide. To restore its binding capacities, the imidazole has to be removed first by using desalting columns. We used molecular weight cut-off spin columns, that retract all molecules above a specific molecular weight and thus let smaller molecules, including the small salt ions pass. With this system the elution buffer can gradually be exchanged against an imidazole free spotting buffer ¹⁾⁴⁾.

Figure 3: Schematic overview of protein purification using gravity flow columns.