The 5 Most Effective Methods of Protein Purification and Expression

Protein is essential to science and every living thing’s survival and biochemical processes. Antibodies found in proteins fend off invading germs and viruses. Researchers are worried about meeting the rising demand for protein. They have redirected their efforts toward synthesizing and generating protein on a massive scale to facilitate even more studies.

Advances in laboratory technology for protein expression and purification services have helped this process. Scientists often produce limited quantities of proteins to research and confirm a protein’s function. In contrast, mass-produced organisms are helpful in areas like vaccine development and the research of antibodies and enzymes. Quality protein yield from custom protein production services requires careful attention to sequencing stages.

1. Extraction

First, you must extract the protein from its source, which may require tissue or cell disruption. To achieve these needs, techniques like freezing and thawing, sonication, homogenization under high pressure, or permeabilization using organic solvents. How delicate the protein is and how robust the cells are will determine the best approach.

After the extraction step, the solvent will include the soluble protein, which can then centrifuge to isolate the protein from the cell membrane, DNA, and other contaminants. Proteins in the solution will begin to be digested by proteases, which are also extracted during the process. Proteolysis is a process that can be slowed by acting rapidly and by keeping the extract at a low temperature.

2. PAGE (PolyAcrylamide Gel Electrophoresis) – Native Gel

Separating a population of DNA or RNA fragments by length, estimating the size of DNA or RNA fragments, or separating proteins by charge are all possible applications of agarose gel electrophoresis in biochemistry and molecular biology, as well as in clinical chemistry for the separation of proteins by charge and size (IEF agarose, essentially size independent). This method makes it possible to categorize molecules. Molecules (like DNA) can be propelled through a gel of agar or polyacrylamide by applying an electric field to the mixture.

One end of the electric field is negatively charged, propelling the molecules through the gel, while the other is positively charged, drawing the molecules along. The molecules to be separated are poured into a gel well. The electrophoresis chamber with the gel inside it is hooked up to a power supply. Electric current slows the movement of larger molecules through the gel while speeding up the movement of smaller ones. The gel shows distinct bands where molecules of varying sizes congregate.

3. Dialysis

One way to do this is by dialysis, which involves swapping the solvent surrounding a protein. The protein solution is often contained within a semipermeable membrane (dialysis bag) and suspended within a greater amount of buffered solution. The membrane needs to allow water and ions through yet block your target protein for this method to work. Buffers and salts are traded back and forth to balance the inside and outside of the membrane.

Hemodialysis and peritoneal dialysis, which remove waste products and excess water from the blood, are the most common types of dialysis used in medicine. Pumping blood through an external filter equipped with a semipermeable membrane flushes away harmful substances and excess fluid. Dialysate travels in the opposite direction of blood flow. Thus, the concentration gradient of solutes is maximized by the counter-current flow of the blood and dialysate.

That allows for more urea and creatinine removal from the blood. Constantly replacing the dialysate ensures that the concentration of undesired solutes is kept low on this side of the membrane. That is because the concentrations of solutes (such as potassium, phosphorus, and urea) are undesirable in the blood but low or absent in the dialysis solution. Minerals like potassium and calcium are present in the dialysis fluid at concentrations comparable to healthy blood.

Peritoneum’s natural semi-permeable membrane filters out waste and excess water from the blood during peritoneal dialysis. Dialysate is a specific dialysis solution introduced within the abdominal cavity to remove waste and excess water from the blood.

4. Centrifugation

Extraction is a purification method that ruptures host-system tissues or cells to release the protein into a solution. The first and most paramount step is to ensure the disordering of the cells where the proteins are found if the organism has not generated the requisite protein into the required solution. Employed procedures include:

i) Sonication repeated freezing and thawing

ii) Homogenization by high pressure or grinding

iii) Permeabilization by detergents or enzymes.

Furthermore, proteases are released after cell lysis, initiating the breakdown of proteins in the solution. The protein purification process should rapidly be completed if there is a reaction with proteolysis, and the extract should be kept cold to control the rate of digestion. Another option is to apply protease inhibitors right before the lysis stages in which the cells are disrupted. Protein stability and host cell viability are considered in the extraction process used for purification. Centrifugation can be used to disentangle soluble proteins from the cell membrane further.

5. Ultracentrifugation

The ultracentrifugation process separates molecules of different densities and masses in a liquid suspension. After putting a protein mixture in a spinning container, each particle will produce a force in the direction of its acceleration, which is directly proportional to its mass. By applying centrifugal force to the liquid, the necessary particles are transported.

The particles encounter a resistance force from the liquid, which propels them forward. On the other hand, massive, small, and heavy particles tend to travel outward when the sample is spun in a centrifuge, as do less massive molecules.

Final Thoughts

Most biochemical studies focus on protein expression and subsequent purification—large production of high-quality protein results from extensive studies into ways to streamline the protein expression process. In addition, advancements in purification techniques have allowed for the quick manufacture of high-quality, pure protein. You can always find a biochemist’s lab that can help you purify and express proteins. Furthermore, there are numerous websites and academic journals available online that provide additional information about protein expression and purification.