Course: SAT 170 Introductory Biotechnology

Unit 5 Learning Module

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Protein Structure and Function (continued)
Protein Expression: Upstream Processing

The process of making protein products for biotechnology begins by expressing a particular protein product; this is also known as upstream processing. Essential to this expression, is isolating a gene that you wish to express and finding a host cell that will be used to express that gene. There are many choices available for selecting a host cell and a few factors must be considered when deciding which host cell to use.

- Bacteria are often a good choice because it is easy to modify their genes. They can also be easily cultured within a short amount of time. However, because they do not possess intercellular organelles like the endoplasmic reticulum and golgi complex (recall from unit 3 that bacteria are prokaryotes which lack such membrane bound organelles), their ability to carry out post-translational modifications is limited.
- Like bacteria, fungi are relatively quick and easy to grow and they are also fairly easy to genetically modify. In addition, since they are eukaryotes they are capable of carrying out the PTM’s described earlier.
- Plants, like fungi, are eukaryotic and so are able to produce proteins and also modify them. They can be grown in large quantities in relatively small amounts of time which contribute to their effectiveness as host cells for protein production.
- Although fungi and plants are eukaryotes and; thus, have similar processes for producing and modifying proteins to animal cells, there are some slight differences in these processes which is why they are not suitable host cells for the production of all proteins. In addition, removal of protein from fungi and plants can be a bit difficult due to the fact that these organisms have a cell wall. For these reasons, mammalian cell cultures and animal bioreactors can be powerful tools. The downside to mammalian cell cultures is that they grow relatively slowly and require more effort to culture, while animal bioreactors are often very difficult to genetically manipulate.

Separation and Analysis: Downstream Processing

Downstream processing refers to the isolation and purification of protein products from their host cells. There are many methods that may be utilized for the separation and analysis of these protein products. In general, the host cells must be lysed (broken open) in order to release the cell contents. Once the cell membrane and cell wall has been ruptured in this way the proteins can be separated from the other cell extracts.

- Filtration is a process that is commonly used to separate these cell extracts based on their size. Various membranes can also be used for filtration. These membranes have pores of various sizes which can allow only materials of a certain size to pass through, while the larger materials are unable to fit through the pores.
- A centrifuge may also be used to pull apart components of a mixture using rotational force. As the centrifuge spins, the heavier cell contents are pulled to the bottom of the containers while the lighter contents remain closer to the top.

See Figure 5: The samples that are ready to be separated can be placed into holders within the centrifuge. These holders or wells will hold the sample stable as the centrifuge spins placing rotational force on the contents of the samples, resulting in their separation from one another.

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- Chromatography laboratory techniques are also used to separate components of a mixture. There are many different types of chromatography, but all types of chromatography rely on what is known as a mobile phase and a stationary phase. The stationary phase is the phase to which a mixture is applied. For example, in column chromatography this is a column that is filled with microscopic resin beads.  A mobile phase, which is some form of liquid, is next added to the stationary phase. The mobile phase will travel through the stationary phase and as it does, it can push or pull the components of the mixture through the stationary phase at different rates depending on their relative size and their solubility in the mobile phase. Thus, each component of the mixture will elute from the column at different times allowing these components to be separated.

See Figure 6: In any chromatography procedure, a mixture (orange dot) is applied to the stationary phase (dark blue). A solvent, or mobile phase (light blue) is then applied to the stationary phase. As the solvent runs through the stationary phase it is able to separate the components (red and yellow dots) of the mixture.

See Figure 7: Column chromatography relies on a stationary phase which consists of resin beads which will allow different compounds of a mixture to be separated from one another as shown here.
- Gel electrophoresis is a process that relies on a gel matrix to separate components of a mixture using an electrical current. Similar to column chromatography, the gel represents a matrix of beads that can act as a molecular sieve separating molecules by size and shape. The force that drives separation is an electrical current. The current will run from a negative electrode to a positive electrode pulling and pushing charged molecules through the gel matrix.

See Figure 8: This is an electrophoresis chamber. The buffer is loaded into the clear chamber and the gel is placed between the red plates in the chamber. The electrodes (red and black) will conduct electrical current that will run through the buffer in which the gel is immersed. Charged particles will be pulled and pushed towards the electrode which exhibits an electrical charge opposite to their own.

See Figure 9: This image shows you step by step how the process of SDS Page is performed. Notice that once the buffer is added and the samples are loaded using a syringe, the chamber is lid is added and an electrical current is applied (the black box shown here is the source of electricity). Once the proteins are separated, the gel can be removed from the chamber.

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- The specific type of chromatography used for protein separation is known as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Before proteins can be separated using SDS-PAGE they are mixed with sodium dodecyl sulfate and heated. The heat causes protein denaturation and the sulfate groups of the sodium dodecyl sulfate attaches to the denatured protein to give it a uniform charge. Once the protein samples are placed in the gel, they will be separated by the size of the protein chain as they travel through the gel. These proteins can then be stained with a protein stain such as Coomassie blue to visualize the banding patterns that result from the separated proteins.

See Figure 10: Different bands will appear on an SDS Page gel depending on how many proteins were present in the original sample from which the proteins were separated. The intensity or width of the band is an indicator of the amount of protein that was present in the original sample.