Protein Purification Techniques

Protein engineering techniques are an essential part of customizing or producing proteins with specific properties which can be applied in various industrial processes. Thus they are crucial to biotechnological research.

However, these methods depend heavily upon being able to isolate and purify the desired proteins so that their physical and chemical properties can be understood, along with their tertiary structures and interactions with ligands and substrates.

Bioprocessing Part 3: Purification

The intensity to which this purification process is pursued depends upon the use to which the protein is to be put. For instance, pharmaceutical and food proteins need to be brought to a high grade of purity, and pass through several sequential steps, as few as possible, since at each step some protein will inevitably be lost.

Purification of protein molecules is simpler than purifying protein complexes.

Protein Purification & Characterization

Step 1: Creating a Crude Protein Extract

Crude extracts of intracellular proteins are prepared by lysing the cell using chemical or mechanical processes. The debris is then removed by centrifugation. The resulting supernatent is a far from pure form, being mixed with many other macro and micromolecules.

Extracellular proteins are obtained by centrifuging the solution and removing the cells. A specific method to obtain a crude extract of thermostable enzymes is to heat the mixture so as to denature other proteins, and then cool it to reform the thermostable proteins of interest, finally centrifuging it to remove the denatured proteins.

Step 2: Intermediate Purification

Salting Out

Proteins in a crude extract are next purified by precipitating them in a highly concentrated salt solution, such as ammonium sulfate. This works on the basis of the lower solubility of protein at high concentrations of salt. However, all proteins do not precipitate at the same concentration of salt, which means salting out also helps to fractionate proteins. It can also be used to concentrate proteins in solution. This step increases the purity three times and 92% of the protein in the solution is recovered.

Dialysis

Proteins are large molecules, and this means the proteins salts will be retained by passing the solution through a semipermeable membrane. Cellulose is a typical dialysis membrane. Dialysis cannot be used to separate proteins of different molecular weights.

Chromatography

Other techniques used to remove salted out proteins include gel exclusion chromatography and filtration. These are now available as preformed kits for many standard proteins, and are often suitable for large-scale processes.

Gel filtration works on the basis of size separation through a column of porous polymer beads, such as dextran or agarose. The large molecules can flow only through the spaces between the beads, while the smaller ones occupy both these spaces and the space inside the beads, slowing them down. Thus the eluent contains molecules emerging in order of their size, from largest to smallest. Reverse-phase or ion-exchange techniques of chromatography are also used, operating on the basis of differential hydrophobic properties and charge respectively. Reversed-phase chromatography may be limited in its application due to possible protein denaturation by organic solvents.

Dialysis and ion-exchange result in a solution which is 9 times as pure, but with only 77% of the original protein being now available. After gel exclusion chromatography, the yield is only 50% but the purity is 100-fold.

Step 3: Final Purification

Affinity Chromatography

This process depends upon using ligands bound to beads which bind specifically to the protein of interest which can then be rinsed out with another solution of free ligands. This results in extremely pure protein samples which have the highest specific activity among all techniques in common use. An example is the purification of concanavalin A using glucose residues attached to beads in a column. The solution is now 3000-fold purer but the yield is only 35% of the original protein.

Polyacrylamide Gel Electrophoresis

Polyacrylamide gel electrophoresis is used to detect the purity of the protein sample after each step based on the size. The net charge on the molecule makes it move down the gel column or sheet in an electric field, making it possible to separate the proteins based on their velocity of migration, which in turn depends upon their charge, as well as the friction and the field strength. The gel acts as a chemically inert and easily formed filter, with protein molecules being almost immobile in the column because they are stuck between the much smaller pores between the molecules of the gel. Initially a series of bands is displayed which represent different proteins in the mixture, which gradually reduce in number till the final step shows only one band.

Immunoblotting

Immunoblotting is another useful technique often combined with affinity chromatography. It uses antibodies for the protein to be isolated as ligands in the column. Sometimes the antibody is attached to isotopes or dyes to label them and make detection easier after separation.

Any chromatographic technique is improved by using pressure to force the solution through a column of finely divided materials, whether charged or ligand-bonded beads. The increased surface area results in greater interaction which pushes up the resolution and speed of the technique. This is referred to as high-performance liquid chromatography (HPLC).

All these steps are included in the ideal scheme, which must concentrate on both yield and purification levels in order to provide adequate amounts of protein to run through an experiment as well as provide sufficient purity to make interpretation relatively straightforward.

References

  1. https://www.thebalance.com/methods-for-protein-purification-375683
  2. https://www.ncbi.nlm.nih.gov/
  3. https://info.gbiosciences.com/blog/bid/154230/what-is-protein-purification-and-what-techniques-are-used-during-this-process
  4. https://www.anu.edu.au/

Further Reading

Last Updated: Jul 19, 2023

Dr. Liji Thomas

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.

Citations

Please use one of the following formats to cite this article in your essay, paper or report:

  • APA

    Thomas, Liji. (2023, July 19). Protein Purification Techniques. News-Medical. Retrieved on October 31, 2024 from https://www.news-medical.net/life-sciences/Protein-Purification-Techniques.aspx.

  • MLA

    Thomas, Liji. "Protein Purification Techniques". News-Medical. 31 October 2024. <https://www.news-medical.net/life-sciences/Protein-Purification-Techniques.aspx>.

  • Chicago

    Thomas, Liji. "Protein Purification Techniques". News-Medical. https://www.news-medical.net/life-sciences/Protein-Purification-Techniques.aspx. (accessed October 31, 2024).

  • Harvard

    Thomas, Liji. 2023. Protein Purification Techniques. News-Medical, viewed 31 October 2024, https://www.news-medical.net/life-sciences/Protein-Purification-Techniques.aspx.

Comments

The opinions expressed here are the views of the writer and do not necessarily reflect the views and opinions of News Medical.
Post a new comment
Post

While we only use edited and approved content for Azthena answers, it may on occasions provide incorrect responses. Please confirm any data provided with the related suppliers or authors. We do not provide medical advice, if you search for medical information you must always consult a medical professional before acting on any information provided.

Your questions, but not your email details will be shared with OpenAI and retained for 30 days in accordance with their privacy principles.

Please do not ask questions that use sensitive or confidential information.

Read the full Terms & Conditions.

You might also like...
Intermittent fasting with protein pacing trumps calorie restriction in gut health and weight loss