Protein engineering techniques are critical for customising or producing proteins with specific properties that you can use in various industrial processes. As a result, they are essential to biotechnological research.
However, these methods rely heavily on the ability to isolate and purify the desired proteins to understand their physical and chemical properties, as well as their tertiary structures and interactions with ligands and substrates.
Significantly, the degree to which this purification process is pursued is determined by the protein’s intended use. Pharmaceutical and food proteins, for example, must be purified to a high degree of purity and pass through as few sequential steps as possible because you will inevitably lose some protein at each step. Below are the sequential techniques involved in protein purification.
Creating a crude protein extract
Crude extracts of intracellular proteins are obtained by lysing the cell using chemical or mechanical processes. Centrifugation is then used to remove the debris using protein production services. The resulting supernatant is far from pure, mixed with other macro and micro molecules.
However, extracellular proteins are extracted by centrifuging the solution and removing the cells. Heat the mixture to denature other proteins and obtain a crude extract of thermostable enzymes. Significantly, cool it down to reform the thermostable proteins of interest before centrifuging it to remove the denatured proteins.
- Salting out: Following that, proteins in a crude extract are purified by precipitating them in a highly concentrated salt solution, such as ammonium sulphate. This works because protein has lower solubility at high salt concentrations. However, because not all proteins precipitate at the same salt concentration, salting out also aids protein fractionation. It is also helpful for concentrating proteins in solution. This step increases the purity threefold, recovering 92 percent of the protein in the solution.
- Dialysis: Because proteins are large molecules, their salts will be retained by passing the solution through a semipermeable membrane. Cellulose is a widely used dialysis membrane material. Significantly, dialysis is unable to separate proteins with different molecular weights.
- Chromatography: Gel exclusion chromatography and filtration are two other methods for removing salted-out proteins. Many standard proteins are now available as preformed kits and frequently suitable for large-scale processes.
Gel filtration works by separating sizes in a column of porous polymer beads, such as dextran or agarose. The large molecules can only flow through the spaces between the beads, whereas the smaller molecules occupy both of these spaces and the space inside the beads, slowing them down. As a result, the element contains molecules that emerge in order of size, from largest to smallest. Reverse-phase or ion-exchange chromatography techniques are also used, which work based on differential hydrophobic properties and charge. Due to the possibility of protein denaturation by organic solvents, reversed-phase chromatography may be limited in its application.
Dialysis and ion exchange produces a nine times purer solution, but only 77 percent of the original protein is now available. The yield is only 50% after gel exclusion chromatography, but the purity is 100-fold.
- Affinity Chromatography: This method relies on ligands bound to beads that bind specifically to the protein of interest before being rinsed with another solution of free ligands. This yields extremely pure protein samples with the highest specific activity of any technique currently in use. One example is purifying concanavalin A using glucose residues attached to beads in a column. Even though the solution is now 3000-fold purer, the yield is only 35% of the original protein.
- Polyacrylamide Gel Electrophoresis: It’s used to determine the purity of a protein sample after each step based on its size. The net charge on the molecule causes it to move down the gel column or sheet in an electric field, allowing the proteins to be separated based on their velocity of migration, which is determined by their charge, friction, and field strength.
Because protein molecules are stuck between the gel’s much smaller pores, the gel functions as a chemically inactive and easily formed filter, with protein molecules almost immobile in the column, initially, a series of bands representing different proteins in the mixture are displayed, which gradually decrease in number until the final step shows only one band.
- Immunoblotting: Immunoblotting is another technique frequently used in conjunction with affinity chromatography. It employs antibodies as ligands in the column to isolate the protein. It is sometimes attached to isotopes or dyes to label the antibody and make detection easier after separation.
- Any chromatographic technique that uses pressure to force a solution through a column of finely divided materials, whether charged or ligand-bonded beads, improves the technique. Significantly, the increased surface area causes more interaction, which increases the technique’s resolution and speed. High-performance liquid chromatography is the term for this (HPLC).
Purified protein reagents are used in a wide range of scientific applications. On an academic scale, numerous reviews detail popular protein production strategies. These strategies are good starting points for producing pure, soluble proteins in milligram quantities. They are used daily by research groups and core facilities professionals with extensive experience with (recombinant) protein expression, purification, and standard characterization techniques.
These are, however, general indications that may necessitate specific adaptations when downstream applications necessitate particular conditions, such as endotoxin-free proteins for animal experiments or nuclease-free proteins for nucleic acid-interaction studies. Similarly, particular proteins have intrinsic biochemical properties that must be considered when designing expression and purification strategies, such as aggregation, disulfide bonds, or a high affinity for nucleic acids.