Dear Praveen,
Here's the summary I give to students when I introduce them to the wonders of protein purification. It's a bit long (so ... not really a summary, LOL) but some of it might help you if you're new to this. Many people don't use removable His-tags as these tags are quite small and mostly do not interfere with crystallisation. However, removing your His-tag safter Ni-NTA might help as sometimes protein molecules aggregate via their His-tags, particularly if a little bit of Ni2+ has leached from the beads/column. You can reduce this by adding 1 mM EDTA to your first dialysis step after Ni-NTA to remove the Ni2+ from your buffer (you can remove the EDTA with a second dialysis step), or by simply removing the His-tags from your protein at this point. Also, sometimes His-tags interfere with crystallisation, so it's best to remove them if you're in doubt. The presence of contaminating proteins can also lead to precipitation after Ni-NTA and your protein might actually become stable if you simply add a second purification step such as ion exchange or size exclusion chromatography. Since you mention you want to carry out structural studies with the protein, you will almost certainly need further purification steps anyway. Some proteins are temperature sensitive and will aggregate if kept too warm or too cold. My current proteins come from thermophilic bacteria and they quickly precipitate if I keep them on ice or at 4 deg C for too long. The best thing is to tweak the lysis/binding buffer to reduce aggregation right from the start rather than after the Ni-NTA step. Things to remember are: During lysis/Ni-NTA: use at least 50 to 100 mM Ni-NTA buffer (TRIS, or HEPES, or phosphate, etc...) at a minimum pH of 7.6 and a maximum pH of 9 (around pH 8 is best unless your protein has a pI of 8, which means your protein would be unstable at pH = 8). Using less that 50 mM might not actually buffer your lysate, which can often be more acidic than you realise after cell lysis. At a pH below 7.6 the binding of his-tagged proteins is reduced, so make sure your buffer is strong enough. use at least 500 mM NaCl in your lysis buffer as most proteins are more soluble in high salt and the non-specific binding of non-his-tagged proteins is reduced. add 2 to 5 mM betamercaptoethanol (BME) to stop contaminating proteins from aggregating or binding to your protein of interest via disulphide bridges, even if your protein doesn't have any disulphide bridges. I prefer not to use DTT at this stage as DTT is a stronger reducing agent than BME and will lead to a brown mess even quicker than BME. you can also add 5-10% glycerol to your lysis buffer as this helps protein solubility/stability. After Ni-NTA: you can dialyse your protein into a different buffer that's better for your protein than the buffer needed for Ni-NTA. 10-20 mM buffer (TRIS, HEPES, etc...) is generally sufficient to buffer the protein solution. Choose a pH that's better for your protein than the pH needed for Ni-NTA. The buffers for Ni-NTA require pH > 7.6 or the His-tags won't bind efficiently to the column/beads. Your protein, however, might be happier at a different pH, so after Ni-NTA choose a pH that is at least 1 point away from your protein's pI (though 1.5 to 2 points is preferable) to make sure your protein is properly charged. Choose to go either up or down the pH scale towards the region closest to pH = 7 to 8 rather than towards the extreme ends of the pH scale (e.g. with pI = 8 go towards pH 6 to 7 rather than pH 9 to 10). use less salt now, 50-150 mM NaCl is usually OK, though some protein are not "happy" with less than 200-300 mM NaCl, particularly those with a pI over 9 or under 5. if your protein has several cysteins that can potentially form disulphide bridges, change the BME to DTT at this stage (1 to 2 mM is generally enough). After your last purification step (e.g after size exclusion chromatography) you won't need DTT or any other reducing agent if your protein doesn't have any cysteins at all and your sample is pure (no other contaminating proteins are present). If you still need a reducing agent, consider TCEP at this final stage, it's probably the most stable BUT it's also VERY expensive, so there's no point in using it in earlier steps. unless you need to remove it because it interferes with your assays, keeping 5-10% glycerol in all your buffers can help with your protein's solubility and will also help if you're planning on freezing it. However, you will generally need to remove the glycerol with a final dialysis step if you plan to use your protein for crystallography as glycerol is a well-known nucleation inhibitor. If you need to freeze your protein for long-term storage, freeze it in small aliquots (50 to 100 micro litres). This way you can thaw as much protein as you need for any given experiment. If you freeze a large volume, then thaw it to remove an aliquot and refreeze the rest of the sample, the sample will often precipitate badly when you thaw it again. So ... freeze small aliquots instead. I hope this helps. Best wishes, Happy Holidays, Merry Christmas (take your pick) and don't work too hard (or at all, if possible, LOL) until the New Year. Tony ------------------------------------------------------ Dr. Antonio Ariza University of Oxford Sir William Dunn School of Pathology South Parks Road Oxford OX1 3RE ________________________________ From: CCP4 bulletin board [[email protected]] on behalf of Praveen Tripathi [[email protected]] Sent: 24 December 2016 10:52 To: [email protected] Subject: [ccp4bb] Need suggestion for protein solubility Dear all, I am graduate student working on a functional protein which i have cloned in pET-28a vector for recombinant protein production in E.coli expression system. The expressed protein is purified on Ni-NTA resins with Imidazole gradient. Surprisingly, i am getting distinct visible white precipitate in pure fractions in eluted fractions itself. Please suggest how to make it soluble or how to prevent the precipitation. On concentrator the precipitate ration is very much increasing. The protein is pure in soluble as well as precipitate. Buffer condition- 50mM Tris(7.5), 500mM NaCl, 10% Glycerol. Elution buffer has varying concentration of imidazole varying from 10mM to 300mM. Any kind of suggestion will be highly appreciated. My project requires structure determination. Thanks in advance. Regards Praveen
