Micro Life Zone
Asked by ghosthunter100 to Arti on 21 May 2012.
Great question! 🙂
I don’t actually isolate enzymes myself, so I can’t really say if it is difficult to do it from personal experience. Other people who I work with take care of this part (biochemistry). I’m more involved with the chemistry side of things, so I make the chemicals that we feed to the enzymes, and also analyse the products that are formed after the enzymes have carried out reactions on them. I have also done a little bit of the actual biological assays with the isolated enzymes, but I mostly stick to the chemistry side rather than biochemistry.
I’ll try to explain the general process for purifying/isolating proteins/enzymes from what I have learnt and also what I have seen people do. Enzymes are types of proteins, so from now on I’ll just use the term protein to keep things simple.
The first thing you would have to do is either get the type of cells (or the whole organism) that have the protein of interest in them naturally (for example, a particular bacteria, or a liver from a dog, or a whole plant or parts of it), or you can produce lots of a protein by expressing it in a different type of cell that it isn’t found in naturally. This is called an ‘expression system’ and ‘expressing’ just means ‘making lots of’ or growing. The type of expression system that you use depends on what your protein is, and is usually a type of bacteria. For cytochrome P450s, people usually use the bacteria Escherichia coli (E. coli) as the expression system because it doesn’t have any P450s of its own. For the bacteria to be able to produce lots of the protein, you need to introduce the DNA that will lead to the production of the protein into the bacteria.
Just a quick run-down on how we go from DNA to proteins in general (this might not make much sense yet if you haven’t ever learnt about this). DNA is first ‘translated’ into RNA – RNA is a molecule like DNA but with some differences (which you can read about if you are interested), so this ‘translation’ step is like the information in DNA being translated into a slightly different language. Then the information in the RNA is ‘transcribed’ into amino acids/proteins. Amino acids are the chemical building blocks that make up proteins. This ‘transcription’ step is like writing the information in the RNA out in a different way.
Once you have the DNA that corresponds to your protein inside the expression system bacteria (if you are interested, you can read about the ways that this can be done), you then need to grow up the bacteria under the appropriate conditions. Then you need to take the bacteria and lyse them, which means break the cells open so that you can get everything out of them. Then you need to separate out all the aqueous solution and water soluble things from anything else (like the pieces of the broken cell), which you can do with a machine called a centrifuge. Centrifuges spin things at very high speeds, so the solids settle at the bottom of the container and the liquid can then be easily separated from them. Once you have the liquid, you need to be able to get your protein out of it.
The actual protein purification part involve a lot of ‘chromatography’, which means separation steps, because you want to separate out your particular protein from all the rest of the things that are also in the solution. Chromatography involves washing your solution through columns full of different solid substances, which interact differently with the different proteins and things in your solution. The different proteins then stick to these different solids (called the ‘stationary phase’) in different ways (some stick more, some less) depending on how much they interact, and so you keep washing the column, the different proteins are separated and can be collected in separate solutions (which are called ‘fractions’). You can separate proteins based on properties such as their size, their ionic charges, or their affinity for particular metal ions, and the type and number of separation steps you would use depends on your protein and how much of it you have. How easily your protein can be purified/isolated will depend on the particular protein.
Once you have collected fractions, you need to find which fractions have your protein in them. Some proteins can be seen, like P450s (which are red) or enzymes called purple acid phosphatases (which are purple, funnily enough!) but some are colourless. In any case, even if you can see a colour, you would want to check if your fractions contain the pure protein or mixtures with other things. You can check this using a process called gel electrophoresis. The main points are that you load some of each of the fractions/solutions you want to test into little wells in a gel. You then put the gel in a tank and have electricity run across the gel, from the bottom where the wells are up to the top of the gel. The electricity separates out proteins based on their size (molecular weight), and you have to stain the gel with some special chemicals so that you can see where your protein bands are along the length of the gel. You can see if your fractions contain only one things or have mixtures, and you also use a reference protein mixture that has several different sized proteins in it as a guide to figure out if the protein(s) you have isolated seem to be the one(s) that you want. This guide is called a ladder and you can see a photo of a gel at this website, where you can also read more about this technique if you are really interested: http://en.wikipedia.org/wiki/SDS-PAGE.
Once you have found the fractions/solutions that contain you pure protein of interest, you would concentrate the solution of a smaller volume, then freeze the concentrated protein solution and store it (usually at low temperature, but how low will depend on the protein) for later use.
I hope this gives you an idea of how difficult protein/enzyme isolation/purification might be!
By BRIDGE8 under license from Mangorolla CIC 2023