CHAPTER 6

GETTING WHAT WE NEED

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INTRODUCTION
Genetic engineering is used to produce therapeutic proteins. To provide a
treatment for diabetes, for example, a recombinant plasmid is engineered to
contain a cloned human insulin gene. Bacteria take up the recombinant plasmid
and express the gene, producing insulin. To date, you have carried out all or
some of these steps using the cloned rfp gene rather than a human gene that
would produce a therapeutic protein.

The final step in the process is to obtain the protein. To do this, bacteria are
treated with a reagent that lyses them (breaks open their cell walls), and
the protein is separated from the cell contents by a method called column
chromatography. (Chromatography is a method for separating similar substances
by dissolving them and then flowing the solution over a material that binds the
substances to different degrees. Column chromatography uses a column packed
with beads coated with the binding material.)

In this chapter, you will complete this final step. You will lyse the bacteria you
transformed in Chapter 5 and then use a column that separates proteins based
on their solubility in water to obtain RFP made by the cloned rfp gene. This same
process would be used to isolate a human therapeutic protein.

CHAPTER 6 GOALS
By the end of this chapter, you will be able to do the following:
• Describe the conditions that are favorable to bacterial growth
• Explain how a protein’s conformation (three-dimensional shape) is related
to its function
• Explain how protein folding (the physical process by which a protein folds
into its characteristic three-dimensional structure, which is essential to its
function) occurs
• Explain how column chromatography separates proteins

WHAT DO YOU ALREADY KNOW?

Discuss the following questions with your partner, and write your ideas in your
notebook. Be prepared to discuss your responses with the class. Don’t worry if
you don’t know all the answers. Discussing these questions will help you think
about what you already know about bacterial growth and proteins.
1. How do bacteria reproduce?
2. Why are proteins sometimes called workhorse molecules?

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 3. How might the conformation (shape or folding) of a protein be important
for its function? Focus on one of the following protein functions: acting as
an enzyme (speeding up reaction rates), transporting molecules, signaling, or
forming structures.

4. A polypeptide is a long linear molecule when it is made, but it immediately

folds into a specific three-dimensional conformation, which we call a
protein. What properties of the amino acids in a protein control the folding
process?

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PRODUCING THE PROTEIN OF INTEREST
After transforming the bacteria so that they contain the plasmid with the gene
of interest, you need the bacteria to get the bacteria to reproduce and then to
express this gene (make the protein that the gene codes for).

BACTERIAL REPRODUCTION
What factors affect bacterial reproduction, which is also called bacterial growth?
The last step in Chapter 5 was to place the bacteria that were transformed with
the pARA-R plasmid into a suspension culture in a shaker flask. The cells were
suspended in a nutrient broth, and the shaking mixed air into the suspension
and prevented the bacteria from settling out of solution, providing conditions
favorable to growth.

CONSIDER: Why might the shaker flask be better at supporting bacterial cell
growth than a plate?

Under optimal conditions, such as those provided by the shaker flask, the growth
of a bacterial population is predictable (see Figure 6.1). The growth occurs in
four distinct phases:
• In the lag phase, there is a zero growth rate. There are no new cells and no
cells dying. The cells adjust to the new conditions, grow larger, and prepare
for cell division.
• In the log phase, there is a logarithmic growth rate. The number of new
cells is greater than the number of cells dying. The cells undergo asexual cell
division and double in number about every 20 minutes (which is the average
doubling time for E. coli; other bacteria have different doubling times). This
phase occurs as long as necessary resources, such as food and oxygen, are
unlimited, and there are no unusual factors that cause cell death.
• In the stationary phase, there is a zero growth rate. The number of new cells
equals the number of cells dying. This phase occurs once resources, such as
food and oxygen, become limited.
• In the death phase, there is a negative growth rate. The number of new cells
is less than the number of cells dying. This phase occurs when resources are
depleted and when toxic waste products build up.

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 Figure 6.1: Change in bacterial growth over time in the shaker flask

Stationary
Log, or
phase
exponential Death, or
growth, logarithmic

Log of numbers of bacteria

phase decline, phase

Lag
phase

0 5 10
Time (hr)

CONSIDER: If the gene of interest is controlled by an operon, such as the

arabinose operon, when is the best time to turn on the gene? Keep in mind:
• Production of the protein takes energy away from the processes of cell
growth and cell division
• A greater number of cells will produce more protein
• Proteins can degrade over time

PROTEIN PURIFICATION
When the transformed bacteria are allowed to grow and multiply numerous
times, they can produce enough therapeutic protein to meet the treatment
needs of patients. However, the therapeutic protein must be purified, which
requires separating it from the other contents of the cell, including other
proteins. (A typical bacterium may contain more than 1,000 different kinds of
protein.) Column chromatography is one method used to separate the proteins.

What physical characteristics of proteins enable them to be separated by column

chromatography? Although all proteins are made up of amino acids, each
kind of protein has a specific function and a specific conformation (shape). The
conformation relates to function because the outside surface of a protein has
specific sites that bind to other molecules. These binding sites allow proteins
to attach to other molecules, which is how proteins can catalyze reactions,
transport molecules, provide a signal, and form structures.

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When a polypeptide is first synthesized, it is a long flexible chain of amino acids,
but it immediately attains its three-dimensional conformation by a process called
protein folding (see Figure 6.2). Once it has been folded, the polypeptide is
called a protein. Protein folding is dependent on the following properties of the
amino acids in the protein:
• Formation of weak noncovalent bonds between positively charged and
negatively charged side chains of amino acids
• The tendency for hydrophobic (water-insoluble) amino acids to be buried
on the inside of the protein away from water and for hydrophilic (water-
soluble) amino acids to be found on the outside of the protein exposed to
water
• Formation of covalent bonds, called disulfide bridges, that occur between
sulfur-containing amino acids

Figure 6.2: Protein folding

Unfolded Folded

CONSIDER: If a mutation changes an amino acid, how might this change affect
protein folding and protein function?

Specific proteins are either hydrophobic or hydrophilic, depending on the

relative amount of hydrophobic and hydrophilic amino acids they contain.
Hydrophobic proteins and hydrophilic proteins can be separated by column
chromatography. In this method, a column is packed
with small beads that are coated with a material (called a
resin) that attracts hydrophobic amino acids, and the
mixture of proteins is dissolved and passed over the
beads. For the hydrophobic amino acids to stick to the
resin, the proteins must be unfolded to expose the Column
hydrophobic amino acids, which tend to be found on the Resin-coated
inside of the protein. Certain salt solutions called buffers beads
(solutions that resist changes in pH) will unfold proteins
to varying degrees.

A series of buffers of different concentrations of salt can

be used to separate many proteins from one another.
For example, to separate the highly hydrophobic protein

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 RFP in a column, a binding buffer is used to unfold all the proteins so that
hydrophobic proteins stick to the resin and hydrophilic proteins pass through
the column. A wash buffer is poured into the column to release moderately
hydrophobic proteins from the resin, and then an elution buffer (used to extract
a substance that is bound to another by washing it with a solution) is poured
into the column to release RFP from the resin. Both the wash and the elution
buffers have a lower salt concentration than the binding buffer and thus cause
bound proteins to fold and begin to cover their hydrophobic amino acids.
Depending on the extent of folding, the proteins are released from the beads.
Figure 6.3 illustrates this process.

Figure 6.3: Using three buffers to separate red fluorescent protein

Binding buffer is Wash buffer is Elution buffer is

passed through passed through passed through
the column the column the column

Binding buffer Wash buffer Elution buffer

Column Column Column

Stop cock

Moderately Red
Hydrophilic
hydrophobic fluorescent
proteins
proteins protein

CONSIDER: If you were trying to use column chromatography to separate

insulin from a mixture of proteins, would you use the same binding, wash,
and elution buffers used for RFP, or would you use buffers with different salt
concentrations? Explain the reasoning for your answer.

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DID YOU KNOW?
Recombinant Proteins

As you have learned, when recombinant proteins are produced for use
as human therapeutics, host cells must be grown in large quantities
so that enough recombinant protein is produced to meet treatment
demand (the needs of patients). The recombinant protein is isolated,
purified, and analyzed for activity and quality before it goes to market.

Producing a protein with the proper order of amino acids isn’t always
the whole story, however. Sometimes further processing or modification
is required to yield an active or fully functioning protein. Many
human proteins are glycosylated, meaning that they have a particular
pattern of sugar molecules linked to them. If a protein is translated
but not correctly glycosylated, it may not function properly. Another
modification involves the addition of a phosphate group—a process
known as phosphorylation. Phosphorylation of a protein can act as
a kind of switch, allowing the protein to become more or less active
by uncovering or covering its binding sites. In addition to saccharide
(sugar) and phosphate groups, other chemical groups may be added to a
protein in order to change its function.

Recombinant proteins for therapeutic use include vaccines (which cause

a body’s immune system to make antibodies that will bind to a bacteria,
or to make a virus to fight a disease), hormones (substances that act as
chemical messengers in the body), monoclonal antibodies (proteins that
bind to substances in the body and are made by clones of a cell formed
in the laboratory), and hematopoietic growth factors (a group of
proteins that promote the growth, differentiation, and activity of blood
cells) for the treatment of diseases including cancer, AIDS, allergies, and
asthma. The number of recombinant proteins has increased greatly in
recent years as the technology used for their production and purification
has advanced.

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LABORATORY

LABORATORY 6: PURIFYING THE

FLUORESCENT PROTEIN
In the previous chapter you transformed bacteria and then selected the bacteria
that had taken up the plasmid of interest (which included the gene for rfp)
by placing the cells on plates that contained LB, ampicillin, and arabinose. The
bacterial colonies that grew on these plates all contained the rfp gene because
the rfp gene was paired with the gene for ampicillin-resistance. Your class or
your teacher then selected one colony (a group of the same kind of organisms
living closely together, usually for mutual benefit), and grew it in a shaker flask
to create a large population of identical cells that all contained the recombinant
plasmid. Near the end of the log phase of bacterial growth, the cells were
given arabinose to turn on the rfp genes so that they would make RFP. In the
first part of this laboratory, you will use a reagent called lysis buffer to lyse, or
break open, the cells. In the second part of this laboratory, you will use column
chromatography to isolate, or purify, the RFP. This is the same process that would
be used to isolate a human therapeutic protein.

BEFORE THE LAB

Discuss the following with your group, and be prepared to share your thoughts
with the class.
1. In column chromatography, how can solutions of different salt
concentrations, which will unfold proteins to varying degrees, be used to help
purify RFP?

2. Read through the Methods sections for Part A (on pages 108 and 109) and for
Part B (on pages 110 through 111) and briefly outline the steps, using words
and a flowchart.

SAFETY: All appropriate safety precautions and attire required for a science
laboratory should be used. Please refer to your teacher’s instructions.

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