Protein Purification Techniques

Protein Purification

Protein purification in biochemistry is crucial for experiments involving proteins, such as structure determination, interaction studies, and activity assays.
In vitro purification allows precise determination of protein structure, avoiding interference from other molecules.
However, in vitro results should be correlated with in vivo methods to account for cellular context.

Protein Sources and Initial Steps

Proteins are purified from various sources (animal, bacterial, plant tissues).
The tissue source should have a relatively high concentration of the target protein.
The first step involves cell lysis to release cellular components.
For intracellular proteins, lysis breaks plasma and cellular membranes.
Typically, tissue is homogenized in a blender with a buffer of specific salt and pH to create a crude cellular extract.

Fractionation and Purification

Purification involves fractionation, column chromatography, and dialysis.
The process aims to remove impurities step by step, resulting in a small amount of highly pure protein.
Protein purification can be time-consuming, taking days, weeks, or months, depending on the protein.
It is essential to research and follow a good, established procedure for the protein of interest.

Exploiting Protein Characteristics for Purification

Protein purification takes advantage of properties like charge, polarity, size, specificity, and solubility.
Ion exchange chromatography, electrophoresis, and isoelectric focusing separate molecules based on charge.
Hydrophobic interactions, reverse phase, and absorption chromatography exploit polarity.
Size-based separation methods include dialysis, filtration, gel electrophoresis, size exclusion chromatography (gel filtration), and centrifugation.
Affinity chromatography is used when a molecule has a natural affinity for a specific group.
Solubility is exploited using ammonium sulfate precipitation after obtaining the crude lysate.

Protein Solubility and Ionic Strength

Ionic strength (I) is defined as $I = \frac{1}{2} \sum<em>{i} c</em>{i}z<em>{i}^{2}$ , where $c</em>{i}$ is the concentration of ion i, and $z_{i}$ is the charge of ion i.
Different proteins have different solubilities at varying ionic strengths or salt concentrations.
For example, hemoglobin is soluble at an ionic strength of 4 but precipitates at 6, while myoglobin remains soluble at 8 but precipitates at 10.
Selective precipitation of proteins is possible due to the narrow solubility ranges of different proteins at different salt concentrations.

Salting In vs. Salting Out

Proteins fold to hide hydrophobic amino acid residues inside and expose polar residues on the outside, making them water-soluble.
Proteins are more stable in solutions with a bit of salt (e.g., sodium, potassium, chloride) than in pure water.
Small amounts of salt increase protein solubility because ions bind to charged residues, counterbalancing these charges.
However, high salt concentrations decrease protein solubility, a process called salting out.
High salt concentrations cause competition between protein and salt for solvation by water molecules, leading to protein precipitation.

Factors Affecting Protein Solubility

Protein solubility depends on salt concentration, solvent polarity, pH, and temperature.
pH affects the ionization of different groups in proteins, altering their charge.
Proteins are least soluble at their isoelectric point (pI), where the net charge is zero.
Selective precipitation of certain proteins is possible by carefully manipulating these properties.
Ammonium sulfate is commonly used to precipitate proteins.
Proteins are precipitated by gradually saturating the solution to 50-55% with ammonium sulfate, which causes proteins with low solubility to precipitate.
After centrifugation, the supernatant is further saturated to 65% to precipitate the protein of interest, which has a solubility of 60%.

Dialysis

Dialysis is used to remove salts that interfere with chromatography and electrophoresis.
Dialysis involves diffusion of molecules across a semi-permeable membrane between the dialysis bag and the surrounding buffer.
The pore size of the dialysis bag is chosen to retain the protein while allowing smaller molecules (like salts) to escape.
The dialysis bag is filled with protein and salt solution and placed in a low ionic strength buffer.
Small molecules dialyze out until equilibrium is reached.
The buffer can be changed multiple times to remove more salt.
Centricons or dialysis cassettes are also used for dialysis, especially for small amounts of proteins.
Centricons have upper and lower chambers separated by a filter membrane, allowing smaller molecules to pass through during centrifugation.

Two-Dimensional Chromatography and Electrophoresis

Two-dimensional techniques provide twice the resolution and separation power of one-dimensional techniques.
In two-dimensional paper chromatography, a mixture of amino acids is separated using two different solvents in perpendicular directions.
First, amino acids are separated in one direction using a polar solvent.
Then, the paper is rotated 90 degrees and separated again using a nonpolar solvent.
Amino acids are visualized after separation by spraying with ninhydrin, which reacts with alpha-amines to form a purple dye.
This approach separates amino acids more effectively than using a single solvent system.

Column Chromatography

Column chromatography is commonly used in biochemistry.
The basic components are the stationary phase (resin) and the mobile phase (buffer).
The stationary phase is packed into a column, and the mobile phase carries the molecules through the stationary phase.
Separation is based on the attraction of molecules to the stationary and mobile phases.
The stationary phase consists of beads or matrix derivatized with different groups (polar, nonpolar, charged).
The resin is mixed with an appropriate buffer before loading into the column and must be kept in equilibrium with the buffer.
The column must never be allowed to dry out, which can cause cracks and require the purification to be restarted.
The column should be kept straight vertically for reproducible results.
After separation, the column can be cleaned, stored in the fridge with a solution to prevent bacteria and fungus growth, and reused.
The effluent fractions are collected and analyzed to identify the protein of interest.
The mobile phase may contain loading buffer and elution buffer with different salt concentrations or pH.

Hydrophobic Interaction Chromatography

Hydrophobic interaction chromatography, also called reverse phase chromatography, uses a resin derivatized with hydrophobic groups (alkane or aromatic groups).
It is used for purifying hydrophobic molecules.
High salt concentrations promote hydrophobic interactions.
A mixture of proteins is loaded onto the hydrophobic resin in high salt, promoting the binding of hydrophobic proteins.
Water-soluble molecules pass through the column.
Proteins are eluted by gradually decreasing the salt concentration.

Protein Purification Profile

A UV detector measures the absorbance of eluting proteins at 280 nm, which is due to aromatic amino acids (tryptophan, tyrosine, and phenylalanine).
This creates a purification profile.
The purification profile plots optical density (absorbance), salt concentration, and enzyme activity against fraction number or time.
For example, in the purification of tyrosine decarboxylase (TDC), proteins are loaded onto the column at high salt concentration.
As proteins elute in different fractions, decreases the salt concentration.
The eluted fractions are collected and assayed for enzymatic activity using a colorimetric reaction.
The intensity of the color change indicates the concentration of the enzyme.

Ion Exchange Chromatography

Ion exchange chromatography includes cation exchange and anion exchange chromatography.
Cation exchange chromatography uses a negatively charged resin to bind positively charged molecules (cations).
Anion exchange chromatography uses a positively charged resin to bind negatively charged molecules (anions).
At a specific pH, proteins have a net charge that determines their binding to the resin.
To elute proteins from the column, change the pH of the eluting buffer, or increase the ionic strength or salt concentration of the buffer.
Changing pH can alter the charge of the protein, causing it to be repelled from the column.
Increasing salt concentration causes competition between the protein and salt ions for binding to the charged resin.
A higher salt concentration is required to elute proteins with greater charge.

Size Exclusion Chromatography

Size exclusion chromatography, also called gel filtration chromatography, separates molecules based on size.
The resin contains hollow beads with different sizes of pores.
Molecules larger than the pore size elute quickly, while smaller molecules penetrate the pores and elute later.
Column length, diameter, and flow rate of the mobile phase affect separation.
The larger the molecule is, the faster goes around the beads without penetrating the beads. These will elute first.
Vice versa, the smaller a molecule is, the more it is able to penetrate the beads and elute later.
In the fractionation profile of superdext 200, the number 200 refers to kDa pore exclusion size, this separation has bovine serum albumin (BSA) of 66 kilodaltons, carbonic anhydrase of 29 kilodaltons, another unknown monomer of g 98, and lysozyme of 14.6 kilodaltons.

Determining Molecular Weight

Size exclusion chromatography can be used to determine the molecular weight of an unknown protein.
The column is calibrated by loading a mixture of proteins with known molecular weights.
The elution volumes of the proteins are plotted against the logarithm of their molecular weights to create a standard curve.
The elution volume of the unknown protein is then used to determine its molecular weight from the standard curve.
The reference standard of cephalodext g 200 has different molecular weight and log mass values to form a calibration line. After the linear regression, it's possible to find the protein of the interest in this column with 200 kilodaltons size pore exclusion.

Affinity Chromatography

Affinity chromatography uses specific protein-ligand interactions for purification.
The stationary phase is derivatized with a ligand that the target protein has a high affinity for.
For instance, a glucose-binding protein can be purified using a resin derivatized with glucose units. Affinity chromatography is specific for bacterial antibodies to increase efficiency during purification process.
Only the protein that binds to the ligand will bind to the column, while everything else goes through.
To elute the protein, a free ligand solution is used.
The free ligand competes with the immobilized ligand for binding to the protein, causing the protein to elute.
The free ligand concentration increases, and it's even more likely the complex between the target binds to a new solution of glucose.
Also useful to establish means with spot test and enzymatic properties or other means to check. Free glucose is loaded to the mixture and only binds to the glucose bonding sites.

Enzyme Assays

If purifying an enzyme, it is useful to establish an enzymatic assay to detect the enzyme in the eluting fractions.
An assay can detect tyrosine decarboxylase (TDC) for enzyme, which will signal fractions based on presence, thus pinpoint locations based on trace lines by decarboxylase activity.
For example, staphylococcal nuclease hydrolyses nucleic acids through the phosphodiester at DNA fragments. Design an assay with pseudo substrates that are then cleaved to show intensity and pH under yellow color.

High-Performance Liquid Chromatography (HPLC)

HPLC is widely used for purifying biological molecules.
Solvents and buffers are pumped through pumps under pressure.
Separation is accomplished under high pressure.

Protein Purification Strategies

If purifying a previously purified protein, consult the literature for an efficient purification protocol.
If purifying a novel protein, start from scratch and determine its amino acid composition, size, isoelectric point, and other physical properties.
Develop a purification scheme, such as lysing cells, ammonium sulfate precipitation, and ion exchange or affinity chromatography.
At each purification step, record the fraction volume, total protein, and activity (total and specific).
While the protein gets pure, the fraction volume goes down, the total protein goes down, total activities go down.

Total vs. Specific Activity

Total activity depends on the total protein present.
Specific activity is a measure of the purity of the enzyme, reflecting the amount of enzyme per milligram of total protein.

Affinity Chromatography Benefits

Affinity chromatography provides the greatest amount of power with the greatest amount of resolution to minimize time/money/headaches. To exemplify, rat liver glucokinase is load and supernate, also with stepwise and affinity chromatography.
Traditional scheme is slower and more time consuming, the stepwise with only three steps increases fold of purification, increases glucokinase, saves yield of the protein sample by 86 percent, and also has benefits by total protein.