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Navigation: Go back to the Protein purification
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Protocols and tips in protein purification or How to purify protein in one day.
General principles of protein purification
Common sense is the best guide in protein purification. For structural studies we should purify protein to a reasonable purity (90+%) with the best possible yield in the shortest possible time and with minimum effort.
Taking all this into consideration, the best possible strategy for protein purification would be one started with affinity or pseudo affinity chromatography (ACh.). This can give you purification folds of tens or even hundreds and results in almost pure protein if the target protein (TP) was properly expressed. An affinity matrix could be a matrix with cross-linked substrate, pseudo substrate or inhibitor. The problem is that we need to create a special matrix and to optimise conditions for chromatography for each protein individually; this may take a lot of time.
The alternative is Tag Affinity Chromatography which is now widely used. The protein gets expressed with a tag attached genetically to it. The most common tags are His6 (fished out on a Ni immobilised column) and GST (fished out on a Glutation column). There are, however, some restrictions to these methods. Firstly, it is not always possible to express protein with the tag on it. Secondly, proteins with tags are useful for many applications, but not for all.
In certain cases pseudo affinity chromatography should be the first chromatographic step in the purification protocol:
- Heparin chromatography should be used for DNA-binding proteins
- Dye chromatography used for NAD/NADP binding proteins
- Protein A chromatography used for antibodies
For purification of the majority of enzymes and other soluble cytoplasmic proteins, we propose Common sense protocol based on using 3 different types of chromatography; separating proteins by their charge (ion exchange chromatography, IEC), hydrophobicity (hydrophobic interaction chromatography, HIC) and size (gel filtration, or size exclusive chromatography, SEC). Common sense dictates the sequence of the steps. It is logical to prepare cell free extract in low salt buffer pH 7-8 and apply it directly on an ion exchange column. After this step it is easy to prepare the sample for hydrophobic chromatography by addition of ammonium sulphate. At this stage an ammonium sulphate cut could also be considered. After hydrophobic chromatography, the protein can be concentrated by precipitation with ammonium sulphate or by a milder method, using concentration by ultrafiltration (pressure unit or spin concentrators) and applying onto a gel filtration column. As a rule, these 3 steps are enough to purify overproduced protein to purity 90+% (this is in cases where the level of target protein (TP) expression is higher than 10% of total cell protein). If the level of TP expression is high (20+%) it may be that only 2 chromatography steps are required to reach an acceptable level of purity (IEC-SEC, HIC-SEC or IEC-HIC). In some cases, especially if the level of TP expression is low, a fourth step should be considered which could be HPLC ion exchange chromatography or low pressure IEC on a different type of matrix at the same or a different pH as that of the first IEC, HPLC hydrophobic chromatography, chromatofocusing or possibly even more exotic solutions. Another problem is that for some proteins hydrophobic chromatography is not suitable as they bind to a hydrophobic matrix irreversibly. To avoid losing the whole pool of protein on a hydrophobic column I suggest taking the less pure sides of the TP peak obtained on an ion exchange column and using them to test HIC compatibility.
About 80% of 180 proteins on my list were successfully purified using the above strategy.
Apart of the using optimal sequence of procedures:
The main tips which help to save time during purification
- Make proper stock solution which would last for long time so you can prepare any buffer solution for your purification in seconds
- Have basic set of columns pre packed and kept in good order
- Preparing cell free extract by sonication divide cell suspension between 2-4 portions and put pieces of ice in to them so you can save time on cooling process
- To remove cell debris apply only 10-15 min centrifugation, the longer spins is not practical because there is no need to remove small particles as they pass the column safely making no harm
- After you apply sample on the column do not wash out unbound material with the starting buffer. The only exception is if TP is going to be eluted from the column very close to the start of the salt gradient. With the beginning of the gradient the unbound material is removed from the column much more effectively than with the starting buffer
- If expression level of TP is higher than 10% in most cases the highest protein peak on the chromatogram is TP. So you can find it by checking protein concentrations in fractions by fast method of Bradford and save time on gel analysis
- If you need to analyse fractions by gel and you have not got pre cast ready to use gels, then start to prepare gel immediately after you have started the chromatography
- Do not wait until all gradient is applied on a column. It is useful to start to analyse protein concentration and activity (if applied) in the fractions after about one third of the gradient have been applied on a column. This is normally position of TP elution if gradient was properly optimised
- If you use gel to find TP after first chromatography, to reveal result quickly stain gel for 5-10 min with the fresh stain and distain it briefly for a few minutes so just to reveal the major bands. As a rule, TP could be identified successfully this way. Combine appropriate fractions and move on for next step leaving gel to be properly stained-distained later.
Important notes to remember before you start purification:
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