What is Protein Purification?

There is an adage in biochemistry: “Never waste pure thoughts on an impure protein” (1). The straightforward definition of protein purification is the isolation of only one type of molecule from the soup of many molecules — but the process itself is rarely so straightforward.

What are the sources of proteins?

The obvious answer is that they come from the cells of an organism. But how can we single out one protein from this complex mixture and isolate it from the rest? This has always been a big question. In the early days of biochemistry, scientists would spend weeks finding the perfect combination of the techniques that will obtain the purified target protein. However, later advances in biology introduced us to recombinant proteins. In a nutshell, these are proteins that are artificially produced in special cells, and, by design, can be produced in much larger quantities.

How do we “artificially” produce proteins in the cells, and which organisms/cells can we use for this?

To explain this better, let’s first go back to the central dogma of biology: DNA is transcribed into RNA, which is translated into protein. If we manage to sneak a piece of DNA into specific cells, we can actually hijack their protein production machinery, and make them synthesize our protein. But which cells can we use? The answer is simple — any type of cell! Every cell has DNA, every cell transcribes its DNA into RNA instructions, and every cell can translate those instructions into proteins. There are many standardized cells used for protein production, varying from simple bacterial cells to actual human cancer cell lines. The list also includes yeast, insect, and mouse cell lines, all specially adapted for protein expression purposes. Each system comes with its own unique set of advantages and disadvantages. Therefore, a cell line must be carefully chosen with the final experiments in mind.

Engineering affinity tags for protein purification

The complex formed between a His tag and the nickel column.

Once we learned how to sneak the desired DNA fragments into cells (and hence, create protein), we realized that we could alter our protein sequences with specialized “tags”. These tags are amino acid sequences added to the ends of recombinant proteins, which let them bind to specific surfaces. If we apply a tag to just our protein, we can very specifically fish it out from the mixture of all other proteins in a single step. These types of purification methods are known as affinity purification methods, as the tags add an affinity to specific types of surfaces. Perhaps the most commonly used tag is a 6 His repeat. This tag adds 6 histidine amino acids to the end of the protein of interest. Histidine has a high affinity for nickel, so a protein tagged in this way will bind to a special nickel-containing resin in a purification column.

Here is a short list of other types of protein purification methods:

Salting Out — is based on differential solubility of proteins in the presence of various salt concentrations.

Reverse-phase chromatography — separates proteins based on their relative hydrophobicity — or how much they “like” to interact with water.

Ion-exchange chromatography — is based on protein surface charge. Columns can be either anion (negatively charged atom) exchange or cation (positively charged atom) exchange.

Size-exclusion chromatography — separates proteins based on their size.

Why is protein purification important?

For many reasons. First of all, we can confirm the amino acid sequence of the protein, and how this relates to other proteins of the same organism and the corresponding protein in other organisms. Additionally, we can study its biochemical activity and function without interference from other molecules, we can determine its 3D structure, and most importantly, we can better understand its implications in human disease.

Links and Citations:

1. Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002. Section 4.1, The Purification of Proteins Is an Essential First Step in Understanding Their Function. Available from: https://www.ncbi.nlm.nih.gov/books/NBK22410/
2. All pictures for this blog were provided by the Wellcome Collection: https://wellcomecollection.org/

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