- People with weak immune systems (AIDS patients, cancer patients, severe asthmatics, etc.)
- Diabetics
- Athletes participating in contact sports or weight training
- Young children
- The elderly
- Persons staying in a health care facility for an extended period of time
- Prisoners or anyone living in confined space with other people.
- Surfers who spend large amounts of time in coastal waters where MRSA is present
In science Protein purification involves a number of processes that aim to isolate the desired protein from a complex mixture. It is necessary in order to be able to characterise the function, structure and interactions of the protein of interest, Biological or microbial cultures are usually the starting material for the process. Cell lysis is the initial step in which the protein is freed from the matrix in which it is confined as the cell membrane is disrupted and its internal contents released into a solution known as crude lysate. Ultracentrifugation is used to purify this mixture which then separates the protein from other components including non-protein constituents of the mixture and finally isolated from other proteins that are present. Fractionates of the various cellular components are produced, fractions include soluble proteins, membrane proteins, nucleic acids and cellular organelles. The proteins from the lysate can be concentrated using precipitation. There are many Different types of chromatography for protein isolation. Protein separation methods make use of difference in molecular weight, chemical properties (such as net charge) and binding affinity of proteins. Different types of gel electrophoresis is utilised (molecular weight and isoelectric point of protein must be known.) to monitor the level of purification and to check the purity. If the protein has distinguishable sprectroscopy features, spectroscopy can be used to monitor level of purification. various enzyme assays used if protein has enzymatic activity. Electrofocusing can be used to isolate proteins according to their charge, all methods are proven and are currently used in labs to separate and examine proteins from each other.
As there are many proteins, they must be extracted from the starting material, which are usually cells; an efficient protocol needs to be developed in order to release the protein by cell disruption. Cell lysis techniques are widely used, this method involves repeated freezing and thawing of tissue or cells containing required the protein.
Homogenization is used for large cells broken by chopping action; this is carried out on muscle tissue as well as plant and animal tissue. Hand homogenization is common as it is where cells are forced through narrow gaps which result in the disruption of cell membrane, for example the liver tissue.
Other cell lysis methods for extracting proteins:
Enzyme digestion – cell wall digested leading to osmotic disruption of cell membrane.
Figure 1:
Figure 1: diagram showing how the cellular membrane can be broken down by using specific enzymes
Osmotic shock lysis – osmotic disruption of cell membrane, this method takes approx. 5 mins.
French press – cells forced through small orifice at very high pressure, 10-30mins, used for plant and bacterial cells
Figure 2:
Figure 2: above diagram shows the cell lysis via forcing cells through a very narrow space under high pressure conditions
Grinding with glass beads, cell walls ripped off by rapid vibration of glass, time: 10-20mins, used for bacteria.
Grinding with alumina / sand is when cell walls ripped off by micro roughness, 5-10mins, again used for bacterial.
Sonication is cell disruption by shear forces and cavitation caused by high-pressure sound waves, 5-10mins used for bacteria.
Figure 3:
Figure 3: diagram illustrating how cells can be broken down with high frequency sound
All these are various cell lysis techniques so that the cell wall or membrane can be broken down in order to reveal the intracellular components for extraction.
Figure 4:
Figure 4: diagram to show the resulting homogenate, or cell lysate. Lysate contains small and large molecules from cell cytosol, such as ribosomes, enzymes abd membrane-bound organelles.
Extraction: homogenization uses ice-cold buffer which is isontonic so there is equal osmotic pressure inside and outside of cell to prevent osmosis which can lead to cell bursting or cell shrinking.
Isolating proteins is precipitate solution with ammonium sulphate, process called salting out. This is carried out by adding increasing amounts of ammonium sulphate followed by collection of the different fractions of the precipitate protein. As salt concentration is increased, more water associates with the ions. The result is less water available to be involved with the salvation layer around protein which therefore exposes hydrophobic patches on protein surface. Proteins then collect and precipitate from solution due to hydrophobic interactions. The advantage of this mthod is it can be performed inexpensively with large volumes. Other forms of precipitation include precipitation by using organic solvents (ethanol or methanol) and using metal salts – at low concentration, called polyvalent metallic ions, most common used: e.g. Ca+, Mg2+.
Protein separation methods are preparative chromatography. There are various types which all make use of different properties of proteins, such as difference in molecular weight, charge. Chromatography involves the passing of a dissolved mixture (‘mobile phase’) through a ‘stationary phase’ – analyte (desired protein) separated from other molecules (other proteins) in the mixture and allows it to be isolated.
Figure 5:
Figure 5: this illustration shows the centrifugation process simplified. After each subsequent spinning of the mixture, a pellet is formed containing the densest particles of the mixture. The supernatant is removed and centrifuged at high speeds to separate out the next heavier particles. This is carried on until the lightest particles form a pellet at the bottom of the tube.
Chromatographic methods
Liquid chromatography has been used for isolating proteins, peptides, and other molecules from complex mixtures. Usually a protein purification protocol contains one or more chromatographic steps. The basic procedure in chromatography is to flow the solution containing the protein through a column packed with various materials. Different proteins interact differently with the column material, and can thus be separated by the time required to pass the column, or the conditions required to elute the protein from the column. Usually proteins are detected as they are coming off the column by their absorbance at 280 nm. Many different chromatographic methods exist:
Separation of Proteins Using Molecular Exclusion Chromatography
This method is basically used to separate macromolecules on the basis of number properties, such as size, charge or affinity for other molecules. Dialysis is the simplest method used for separating small molecules from proteins through a semi-permeable membrane. Pores in the membrane allow molecules up to 10KDa to pass through, but larger molecules are retained. Small molecules therefore move out into the buffer, the buffer is placed in the membrane.
Gel Filtration is also used to separate substances according to molecular size; the method involves a column filled with very small beads of an insoluble but hydrated carbohydrate polymer, which is suspended in buffer. When a mixture of molecules are applied to the top of the column, followed by buffer solution, the molecules move down the column at different rates, depending on there sizes they can be collected as they emerge from the bottom of the column, small molecules are able to enter the beads of the gel.
Ion Exchange Chromatography
Proteins can be separated on the basis of their net charge by ion- exchange chromatography; a protein with a net positive charge will normally bind to a solid phase containing negatively charge carboxyl ate groups. On the other hand if the solid phase contains positively charged tertiary amino groups, then negatively charged proteins will bind but proteins with a positive charge will not. Chemically modified cellulose is often used as solid phases for protein separation.
In conclusion, the whole process of protein isolation and purification involves 5 main steps. Firstly, the protein has to be extracted from cells, by breaking up cells (homogenisation) and secondly extraction from other cell components (centrifugation). Extraction from other cellular proteins (ultracentrifugation). Centrifugation separates components based on their densities, thirdly, precipitation steps are (soluble proteins form precipitate in solution) to recover protein from extract mixture. fourthly, use of chromatography to further separate the target protein from bulk protein in order to check the purity and further purifying by use of electrophoresis, assay procedures (based on enzymatic activity or another easily montiorable property specific to the protein. Therefore in the healthcare area where associated infections are needed to be discovered, the isolation of proteins is important, to detect and treat these infections.
There fore in a fight against Healthcare associated infection, should be made more aware and took more seriously, as it is very important for patients to stay safe when seeking treatment from healthcare practises. Strict rules in hygiene should be followed when staying or receiving treatment and the public should be aware of the treatment and consequences and what they are going to receive.
Overall the detection procedures used are very good and can separated proteins so that they can be examined and be identified which protein it is.
References:
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Voet D, Voet JG. (2004). Biochemistry Vol 1 3rd ed. Wiley: Hoboken, NJ
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Lodish H, Berk A, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipurksy SL, Darnell J. (2004). Molecular Cell Biology 5th ed. WH Freeman and Company: New York, NY
-
Clive Dennison.. A Guide to Protein Isolation. Science,2003.
-
James B. Sumner, Karl Myrbäck, 1952. The enzymes: chemistry and mechanism of action.
-
David Rickwood, 1984, Centrifugation: a practical approach. Technology & Engineering
-
Laszlo Lorand, Gertrude E. Perlmann, Sidney P. Colowick. Proteolytic Enzymes. Science, 1981
-
Nicholas Catsimpoolas, 1975. Method of Protein Separation. Science.
