8.3: Pre Lab Activity- Think Pair Share-Discuss What You Know

Prior to the lab period you should read the material, take some notes on your thoughts on the following questions (Think). In lab, you will be asked to discuss with your partner your thoughts and hear theirs (Pair). Take additional notes on new insights during this small discussion. Finally, you may be asked to discuss your ideas with the class (Share). It is not expected that you know all the answers before the class discussion but by the end of the activity, you should have the questions answered completely. 

1. What is the term for bacterial reproduction? Describe this process.
2. What roles do proteins play within cells? 
3. What happens to protein function if a protein loses, or never correctly achieves, the prescribed conformation?
4. How does the order of amino acids relate to protein conformation and thus protein function?
5. Since we can control when to “turn on” or express our gene, when is the best time to do so? To help you decide ask yourself:
   1. Would producing protein take energy?
   2. Would a greater number of cells produce more of your protein of interest than fewer cells?
   3. Can proteins degrade if left too long?

As you read through the procedures for bacterial lysis and column chromatography, create a flow chart using words and pictures to describe the process.  Compare your flow charts with each other prior to beginning lab. Note any differences and attempt to resolve which flow chart(s) has/have more accurate information. Adjust your won flow chart as necessary.

6. Compare your flow charts with each other prior to beginning lab. Note any differences and attempt to resolve which flow chart(s) has/have more accurate information. Adjust your own flow chart as necessary.

Protein Purification

Transformed bacteria can multiply in culture and produce the protein of interest. If this protein is to be used therapeutically, it will need to be purified.  This means that other cellular components, including other proteins, must be separated from your protein. Column chromatography is a common method to separate proteins. 

Figure 14.2. Protein

Proteins are made of amino acids. Individual amino acids have different properties such as hydrophobicity (water-hating) or hydrophilicity (liking water), ionic charge, or the ability to form weak or strong bonds with other amino acids. When a protein is first made in a cell, it is a long chain of amino acids in an order determined by the gene.  The order of the amino acids in a protein determines how the chain will fold to produce a three dimensional protein conformation (See Figure 14.2). This specific conformation will have different amino acids interacting with each other in specific ways.  Amino acids facing the environment in a folded protein can interact with other molecules. Specific groups of amino acids near each other can form binding sites to interact with other specific molecules. Overall, these relationships determine protein structure and thus protein function. Imagine proteins involved in enzymatic reactions, as channels in membranes, in transporting other molecules, or for binding DNA. These proteins all have very specific binding interactions determined by amino acids in specific locations in a folded protein.

Question: If individual amino acids are swapped or deleted in an amino acid chain, do you imagine this would affect the function of a protein?

The rules for protein folding are not perfectly understood and is an area of active scientific investigation. However, a few basic rules have been discovered. Factors that cause proteins to fold in specific ways include:

1. Weak bonds will form between amino acids with a negative and a positive charge. 

2. Strong (covalent) bonds will form between sulfur-containing amino acids. These are called disulfide bridges. 
3. Hydrophilic amino acids locate to the outer surfaces of proteins because they interact with the cell environment, which is mostly water. Hydrophobic amino acids hide on the inside of proteins or embed within cell membranes to avoid contact with the water in the environment. 

 

Figure 14.3. An image of a column with resin beads and a collection tube

Depending on the content of amino acids in a specific protein, overall it will take on a hydrophobic or hydrophilic character. Column chromatography can separate hydrophilic and hydrophobic proteins from the rest of the cell contents.  Small beads coated with a material called a resin are packed into a column. The resin attracts proteins which will bind to the resin as other cell contents flow past. For hydrophobic proteins to stick, they must be treated to expose the typically buried hydrophobic amino acids. Buffer solutions can be used to cause proteins to denature (unfold) and expose the amino acids that will be attracted to the resin. 

Different buffers are passed over the column in an order determined to best separate the proteins of interest from the rest of the cell contents. Figure 14.4 shows three solutions used to separate green fluorescent protein from the rest of the cell.  The binding buffer denatures proteins so that the hydrophobic amino acids stick to the resin. The wash buffer removes loosely adherent proteins and material from the column leaving the more strongly attached protein of interest. Finally, the elution buffer, which has a low buffer concentration, causes the protein to begin to refold to hide the hydrophobic amino acids which releases the protein from the resin coated beads in the column. The portion or fraction of fluid exiting the column that contains your protein can be captured in a container and saved. DOES SIZE PLAY A ROLE? DO WE SAY ANYTHING ABOUT IT?

Figure 14.4. Separation of green fluorescent protein by hydrophobicity using column chromatography

Question: Do you believe that all types of protein would use the same types of resin-coated beads and the same types of buffers to become purified? Explain your answer.