Many biochemists purify their beloved proteins, whether at small or large scale and for different reasons (identification, enzymatic reactions, characterization etc.). There are several rules and tips that can facilitate the purification process and increase your chances to have a pure protein in your freezer at the end of the day.
Predict and plan
The first step is to perform some basic bioinformatics analysis. The most important ones are those that relate to the physio-chemical properties of your protein such as amino acid composition, extinction coefficient, secondary structure prediction and motif prediction (see here for a comprehensive list of bioinformatics tools). Another good prediction website worth mentioning is Xtalpred, which acts as a meta server for the most basic protein analysis.
Once you gathered some basic knowledge about the protein, you can now assess:
- Membrane or cytosolic
- Number of cystienes
- Predicted pI
- Expected extinction coefficient
- Motif or structural domain which necessitates the addition of ions or co-factors
All the above will affect your expression system approach and your subsequent purification process. Plan ahead when to do an expression tests (different hosts, different induction media, different temperature and induction times). Evaluate how much protein you should require and plan your up-scaling expression accordingly. Check to see that you have the required materials (DNAse, protease inhibitors, columns and FPLC reserved) and prepare your schedule (you should look into Labguru, a web-based laboratory management system, that can help you manage your experiments).
Expressing your protein – making the most of every ml
This is one of the crucial steps in a succesfull purification scheme – you can do 5 purification steps and still be left with a dirty prep or do just 2 steps and get 95% and higher purity level. It is THAT crucial. Getting protein highly expressed in E. Coli is not always trivial but with some initial screening you might struck a successful induction setting (see my earlier post on how to improve recombinant protein expression). What settings should be checked?
- Media – Don’t settle only for LB and IPTG induction; you should try the Autoinduction media, developed by William Studier, which can boost protein expression between 3-10 fold, depending on the protein.
- Temperatures & induction time – Check several temperature points: 18, 27, 30 and 37 degrees Celsius, while varying the induction time appropriately. Meaning, the lower the temperature the longer induction time (we routinely induce proteins at 20 or 27 degrees over weekend). Start with an overnight and modify accordingly.
- Strains – Try inducing with Rosetta, Codon+, Tuner and Rosetta-Gami strains.
All the above settings should be tested at small scale (i.e. 50ml culture) and once you found a successful combination go ahead and up scale. And when I talk about up scaling I am talking about a minimum of 4L culture or higher. You might be surprised at this volume scale, but it is better to get a bucket loads of protein and then throw away major chunks as you cut out dirty fractions. No problem to start with 200mg of protein and then be left with 10mg of super pure protein.
Breaking cells or cell lysis
Assuming most will purify a recombinant protein, your protein will be expected to contain an N or C-terminal tag.
- When breaking cells try to use five volumes of lysis buffer per one gram of cell pellet.
- I found it best to redissolve the frozen pellet with the aid of a stirrer – this way you can continue preparing for the coming purification while the pellet slowly dissolves into the lysis buffer
- If you’re not purifying a membrane protein you can add 0.02% Triton X-100 to aid with the lysis of the bacteria.
- If you’re using His tag, don’t forger to add 10-20 mM Imidzole to the buffer
- Use ice all the times! The sample should be at 4°C all the time.
- Pre-chill the French press in ice about 10 minutes before intial lysis. If you’re having many samples you might want to chill it again for 5-10 minutes per several samples to keep it cold.
Initial separation and affinity chromatography
- Centrifugation at 45,000 rpm for 30-45 minutes is sufficient for most proteins (you can live also with half of it using SS-34 rotor at 20,000 rpm for 2 hours).
- If using a dripping column, initially pack beads that will be sufficient to ~10mg protein/1L of induction. After the first purification it will be easier for you to adjust the amount of beads to get the best purification without getting too much junk.
- Remember that in most cases the affinity chromatography step will determine the fate of your sample in terms of purity levels. Do your best to calibrate this step to the optimal setting possible so you get the protein at the highest purity level. From my own experience a dirty sample post affinity will not improve tremendously after using other columns (again, in most cases).
- Perform extensive washes, especially if working with dripping columns, 20 times the amount of beads volume (20 CV), varying the amount of salts and Imidazole.
- Elution should be conducted at a relative small flow rate to get the sample concentrated at the end of this step; in certain cases you’d want to increase the flow rate to avoid high concentration elution in case the protein is prone for aggregation.
- Evaluate your sample purity through an SDS-PAGE analysis. In case you find it hard to identify your protein do a western blot for verification (blot directly from the coomassie-stained gel).
- Depending on the purity level required and the observed purity of the sample, assess whether you want to do a step of ion-exchange or size exclusion.
- In case you plan on doing a step of ion-exchange, you might consider eluting your protein with low salt concentration (maximum 50 mM ion strength) and continue directly with your post-affinity elution into the ion-exchange column.
To be continued…