Team:Freiburg/Project/Methods/ProtPur
The biochemistry behind protein purification
Cell-free protein expression is a versatile tool for creating 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 thorougly. So we decided to use conventional expression in Escherichia coli to reliably get bigger amounts of protein for testing and optimizing additional to cell-free protein expression.
Using the natural expression machinery: Growing cells
The genetic information for our antigens was transformed into the bacteria we used (see: strains we used). As production of alien proteins (heterologous expression) causes severe stress in the cells, it is reduced by use of the lac-repression system. So only after addition of the non-degradable allolactose analogon IPTG (Isopropylthiogalactopyranosid) the lac-repressor dissociates from its binding site and gives way for transcription. To achieve fast expression the viral T7 RNA polymerase is used instead of endogenous polymerases.
To yield optimal protein concentrations each expression system has first to be optimized in terms of temperature and IPTG-concentration. 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 workflow cells are first grown to an optical thickness of 0.5 before they are induced by IPTG. This is done to ascertain that there are enough cells to overcome the stress caused by induction. After a certain expression time (2, 4 or 8 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 directly degraded by proteases. These proteins don't 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 PSMF, a small organic molecule that specifically inhibits serine proteases, by adding it to the solution just before cell lysis 3). That was done 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 carried out. For this second step higher accelerations were needed to pellet cell organelles and membranes. Most of the target proteins can be found in the pellet (inclusion bodies) as well as in the supernatant (soluble fraction) but only the soluble fraction was used for further purification.
Create a useable protein solution
Even after centrifugation the protein remains far from pure. To specifically address the protein of interest an affinity purification method best suited. Therefore a decahistidine tag is fused to the protein whose interaction with divalent cations as nickel allows for selective binding. 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 eluated with imidazol that competes for the binding to the cation. Despite having a much purer antigen solution, the eluate contains a high concentration of imidazole. Our aim is to adhere the expressed proteins to a glass slide via specific interactions. Since 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, we are able to specifically bind the antigen onto the slide. To restore its binding capacities the imidazole has first to be removed by using desalting columns. We used molecular weight cutoff spin columns, that retract all molecules above a specific molecular weight and thus let pass the small salt ions. With this system the eluation buffer can gradually be exchanged against a imidazol free spotting buffer 4)5).
