9:Purification of protein based pharmaceutical II Flashcards
Explain size-exclusion chromatography
Can also be known as gel filtration chromatography or molecular sieve chromatography.
It consists of fine, porous beads such as dextran polymers, agarose and polyacrylamides. As the solution passes through the beads, proteins that are too large to enter the pores are removed and smaller molecules may be retarded depending on their size.
List the characteristics that proteins are purified and identify the type of chromatography that is used for each characteristic
Size - Size-exclusion chromatography
Charge - Ion exchange chromatography
Hydrophobicity - Hydrophobic chromatography
Binding affinity - Affinity chromatography
Explain ion exchange chromatography
Consists of beads with positive or negative charge. Proteins are therefore separated according to the arrangement of charges on their surface. Bound proteins are removed by gradually adding excess salt solution.
Explain hydrophobic interaction chromatography.
Separates proteins based on the surface hydrophobicity of the protein. The matrix contains hydrophobic groups that will interact with hydrophobic regions of proteins. This matrix is bound to a column in a high ionic strength buffer. The hydration of the salt ions attracts water molecules away from the protein, hence increasing the hydrophobicity of the proteins. These can then be removes by decreasing the ionic strength with the addition of a solvent of detergent.
Explain affinity chromatography
A mixture of proteins are added to a column consisting of a polymer-bound ligand specific to the protein of interest. Unwanted proteins are then washed out the through column with water. A solution of ligand is then added to the column, removing the proteins of interest with it. The ligand can be an antibody, protein or a drug.
Explain High performance(pressure) liquid chromatography (HPLC)
HPLC consists of small bead particle size for superior resolution and an increased flow rate for improved fractionation speed. It is more expensive and low volume, but yields high value proteins. It is often used as a preparative step in industrial scale purification of insulin and interleukin-2.
Explain UV-absorption as a technique for protein quantification
Contains aromatic ring structures of tryptophan and tyrosine, but it is limited by the content of these residues. It has a sensitivity of approx 10ug/mL. The peptide bonds have an absorbance of 205nm and the side chain has an absorbance of 280nm.
Explain Colorimetric assays as a technique for protein quantification
Lowry method - copper protein complexes reduced by Folin’s reagent. It measures absorbance at 750nm with a sensitivity of 10ug/mL
Bradford method - Utilises the principle of absorbance change consequent on protein dye binding. It measures absorbance at 595nm with a sensitivity of 0.5ug/mL
By constructing a standard curve of the absorbance vs concentration to determine the sample concentration that is read in the linear region of the graph.
Explain Enzyme-immunoassay as a technique for protein quantification and explain how it is done.
Developed using the specificity of antibodies and the sensitivity of enzyme assays. It is preserved in the ELISA (Enzyme linked immunosorbent assay) technique. It is measure using a specialised spectrophotometer called the ELISA reader.
Method;
1)Coat surface with the sample (Antigens)
2) Block unoccupied sites with a non-specific protein.
3) Incubate with primary antibody again the specific antigen.
4) Incubate with antibody-enzyme complex that binds with the primary antibody
5) Add substrate
6) Formation of coloured product indicates the presence of the specific antigen.
Describe how protein purification can be monitored.
The SDS-PAGE can be used to quantitatively evaluate protein purification by monitoring the purification procedure at each stage, the protein is pure one only one band is detected on the gel.
Also, other methods can evaluate purification by the function of the protein using an enzyme quantified by specific activity and functional assays (Hormones, transport proteins and binding proteins)
Explain electrophoresis.
Electrophoresis separates proteins by their charge and size. proteins are chains of charged amino acids, therefore they can be separated by its charge. However, this can be opposed by the viscosity and pore size of the medium used, in which case it can be separated on size.
This method is not commonly used as a preparative method, but is commonly used as an analytical technique to approximate the MW, number of proteins or the degree of purity of a sample.
What is isoelectric focusing and how is it done?
IEF separates proteins on the basis of their relative contents of acidic and basic residues.
This is done by forming a gel with a pH gradient (Ampholytes) and then conducting electrophoresis. Proteins migrate through the gel until they reach a pH that is equal to its own isoelectric point (pI). At this point the net charge on the protein is zero and it stops migrating.
Explain 2D Electrophoresis.
2D electrophoresis combines IEF with SDS-PAGE. The first dimension, horizontal, separates the proteins by IEF and then second dimension, vertical, separates the proteins by SDS-PAGE. This yields a very high resolution preparation.
Explain immunoblotting
Immunoblotting is a technique used to detect a specific protein in a mixture of proteins. This is done by:
1) Transferring proteins from the SDS gel to a porous membrane (blotting)
2) Probe the blot with a primary antibody specific for the protein of interest
3) Probe the blot with an enzyme-conjugated secondary antibody specific for the primary antibody.
4) Add the enzyme substrate and visualise (Semiquantitative)
What are the 6 mechanistic classes of proteolytic degradation? and what do they do?
- Serine proteases I (Mammalian)
- Serine proteases II (Bacterial)
- Metalloproteases I (Mammalian)
- Metalloproteases II (Bacterial)
- Cysteine proteases
- Aspartic proteases
These classes are chemical processes that minimise the processing time and allow it to occur at low temperatures.
Protease inhibitors include;
- Benzamidine: Inhibits serine proteases
- Pepstatin A: Inhibits aspartic proteases
- EDTA: Inhibits metalloproteases.
