QuickTip #9: Purifying a His-tagged protein with low expression level

Do you find yourself having to deal with a very low yield of your target protein after the expression? Are you using a His-tag? Here are some ideas to help you with your purification.

If you can change your expression system to improve the yield that is of course the best.

It can be tough to load a large volume of cell culture media onto a column, particularly if the volume of the cell culture is much greater than that of the column. It will easily clog, even if you carry out thorough sample pre-treatment before loading. Doing a batch purification is one solution, if you don't have proteases in your sample (since these may destroy your target protein as the purification will take longer time). After that, pack the resin into a column, wash and elute, preferably using a linear gradient, especially if it is the first time you are purifying this His-tagged protein.

You will be loading a lot of impurities onto the resin and two things can help improve the situation. First, change to a Co-charged resin that in most cases gives an eluted His-tagged protein with higher purity. Try for example Bio-Works WorkBeads 40 Co-NTA (https://www.bio-works.com/product/imac-resin/workbeads-nta). Secondly, optimize the concentration of imidazole in the sample and starting/washing buffer(s). Usually 10 mM imidazole is recommended, but the higher a concentration that can be used without losing binding capacity the better the purity of your target protein.

Do you have any DTT and/or EDTA in your cell culture medium and buffers? This will cause problems in binding to, for example, HisTrap as these additives will strip off the Ni-ions from the resin. In this case use a resin that is resistant to these additives, such as for example WorkBeads NiMAC (https://www.bio-works.com/product/imac-resin/workbeads-nimac) which is also available in prepacked BabyBio NiMAC 1 ml and 5 ml columns (https://www.bio-works.com/product/imac-resin/babybio-nimac).

 

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The Bio-Works Team

Posted by The Bio-Works Team

The Bio-Works staff are seasoned and enthusiastic (yes, even a bit nerdy) experts in purification resins, methods and strategies with many years of experience of the ins and outs of this field. We love our job and we’d love to help you do yours by sharing some good solid facts, principles and best practice. In short – “purification made simple”.