8.2: Introduction

Proteins are biological molecules made of chains of amino acids that take on a three-dimensional shape called a conformation. A specific conformation is achieved by folding of the amino acid chain in a way that the protein can perform a specific job or function for the cell. If the protein does not fold in the specific way, it cannot do the job it otherwise would. Proteins can be purified and used as therapeutic agents (drugs) to treat patients with specific conditions. Because purifying specific human proteins can be difficult or impossible from human or animal tissues, we can employ cells such as bacteria like E. coli to do the job.  These cells are genetically engineered to make the proteins of interest in large amounts that are more easily purified. 

An example of a therapeutic protein is insulin, used to treat diabetes.  This was the first genetically engineered protein to be introduced as a therapeutic agent. The gene for human insulin was engineered into a plasmid and this recombinant plasmid was introduced into E.coli. The bacterial cells made insulin which was purified to be used for treating diabetes. 

Similarly, green fluorescent protein (gfp) can be produced and purified. Prior to this lab, the gfp gene was introduced by the process of genetic engineering into a recombinant plasmid capable of allowing E. coli to produce protein from the genetic instructions. Producing protein from a gene is termed expression. This recombinant plasmid was introduced into E. coli using the process of transformation.  This laboratory uses the process of obtaining purified green fluorescent protein to model how therapeutic proteins are purified for human treatments. 

You will be provided a culture of bacteria that have been grown and induced (stimulated) to produce gfp. You will treat the bacteria with a solution that will lyse (break open) the cells to release the protein. Then you will employ the process of column chromatography, the focus of this laboratory. Column chromatography is a method of allowing a solution to flow over a substance that will selectively bind and separate the components of the solution. In this case, tiny beads are packed into a tube-like column and the solution obtained through bacterial lysis is allowed to flow over the beads. As you pass other solutions over the column, you will be able to collect some of the solution flowing through the beads which will contain a much more concentrated and pure gfp. 

Growing Bacterial Cells

The pattern of growth for bacterial cells is well understood.  In the laboratory setting, the goal is to grow cells and have them produce the protein we are interested in purifying. When using optimal conditions to grow E. coli, four phases of growth will occur (see Figure 14.1).

1. During lag phase no cell division occurs. Cells are preparing to divide by making new enzymes and proteins as well as copies of their DNA. Cells enlarge. 


   

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2. In the  log phase, a doubling of the population is occurring. This is termed logarithmic growth.  Cells undergo binary fission (cells divide in half) approximately every 20 minutes for E. coli under optimal conditions. Other types of bacteria, and E. coli under less than ideal conditions, will divide at different rates. 
3. The stationary phase occurs after the log phase when there is no overall change in the population size. During this period, cell division is equivalent to cell death and happens as resources such as nutrients and oxygen are depleted and waste products are building up in the environment.

During the decline or death phase of a bacterial culture, cells are dying faster than they replicate. The cell population is decreasing. This occurs as waste builds further and the food supply is exhausted.