BITC1311 Intro to Biotechnology
Chapter 4. Proteins as Products
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Thieman & Palladino Guided Notes
What are some examples of enzymes that have a “depolymerizing” activity that are useful in the consumer
world? Describe some of their their industrial applications. (Table 4.2)
What are some disadvantages to the use of insulin harvested from pigs? How is it produced by DNA
technology?
What are some other examples of an enzyme or protein hormone replacement therapeutic that is produced by
DNA technology? Name the diseases that are treated by these recombinant proteins. (Table 4.1)
In what ways do industrial processes benefit from the use of recombinant enzymes? Cite some specific
examples of industrial processes that are benefiting in these ways from DNA technology.
What are the 4 levels of structure that proteins are organized in?
a. What type of atoms within the protein interact in a hydrogen bond to fold the polypeptide backbone into
either an alpha helix or a beta sheet?
b. What effect does heat and changes of pH have on these hydrogen bonds?
Some proteins are post-translationally modified by glycosylation.
a. What happens to a protein that is undergoing “glycosylation”?
b. At what subcellular site does glycosylation occur?
c. What are some purposes for this glycosylation step?
There are protein engineering strategies that allow scientists to alter the primary structure of proteins. Describe
how this directed molecular evolution technology can accomplish this goal.
Describe some of the general processes involved in upstream and downstream processing of recombinant
proteins.
Make a table listing the different types of recombinant hosts used in the biotechnology industry. In your table,
list some advantages and some disadvantages to the use of each of these hosts.
Sometimes the level of expression of a recombinant can be boosted to over 60% of the total protein in the
recombinant host. Describe how this high level of expression can be achieved. Can you think of why an
inducible expression system might be of an advantage?
How do the introns found in eukaryotic genes pose a problem in expression of these genes in E. coli? How
can this problem be overcome?
What is a possible advantage for expressing a gene as a fusion protein? What is a disadvantage of this
approach?
What is a possible advantage and a possible disadvantage of genes being expressed as an inclusion body in a
recombinant host?
What are 3 parameters that are monitored in a bioreactor during a fermentation and why?
What is the level of purity that the FDA requires of recombinant protein products being used medicinally?
a. Make a table of the 3 general steps in harvesting all types of recombinant proteins from a bioreactor?
b. List the different techniques that can be used to accomplish each of the steps listed in your table.
Describe 4 types of chromatography that are commonly used to purify proteins and describe what must be done
to elute proteins from such columns. What role does a “ligand” have in this process?
How does HPLC differ from the 4 types of chromatography that are used to purify proteins in industrial
processes? How are they the same?
Electrophoresis and HPLC are used to analyze purity of enzyme preparations.
a. How are samples prepared for analysis by these two methods?
b. What is the principle of separation of proteins by each of these two methods?
c. How are the proteins detected by each of these methods?
d. Can active protein be recovered by these methods? Explain your reasoning.
Explain how proteins are lyophilized and what the advantage is of this process.
What 2 techniques can be used to determine and/or verify the correct structure of a protein? Describe how
these techniques work.
Describe a situation in which a genetic disease cannot be characterized using genomic techniques and DNA
sequencing, but can be discovered through proteomic techniques.
a. Why is the technology required in the field of proteomics so complex?
b. Describe how a “protein chip” can be constructed for high through-put analysis of proteins.
c. How will proteomic research help to further to goals of the Human Genome Project?