Biomedical Engineering Reference
In-Depth Information
Animal cell culture is required when posttranslational modifications are essential.
Mammalian cells offer the highest degree of fidelity to the authentic natural product. Insect
cell systems potentially offer high expression levels and greater safety than mammalian cells
at the cost of a potential decrease in the fidelity of posttranslational processing. Transgenic
animals are good alternatives for complex proteins requiring extensive posttranslational pro-
cessing. Transgenic plants and plant cell cultures are emerging as production systems for high-
volume protein products. Plants arewell suited to produce proteins for oral or topical delivery.
The vector must be designed to optimize a desired process. Factors to be considered
include vector copy number, promoter strength and regulation, the use of fusion proteins,
signal sequences and secretion, genes providing selective pressure, and elements enhancing
the accuracy of vector partitioning.
The engineer must be aware of the regulatory constraints on the release of cells with
recombinant DNA. These are particularly relevant in plant design, where guidelines for phys-
ical containment must be met. Deliberate release of genetically modified cells is possible, but
extensive documentation will be required. Two increasingly important applications of genetic
engineering aremetabolic or pathway engineering for the productionor destruction of nonpro-
teins and protein engineering for the production of novel or specifically modified proteins.
Further Reading
Shuler, M.L., Kargi, F.
Bioprocess Engineering e Basic Concepts
, Prentice Hall, Upper Saddle River, NJ, 2006.
Carroll, S.B., Grenier, J., Weatherbee, S.D.
From DNA to Diversity: Molecular Genetics and the Evolution of Animal
Design
. 2nd ed. Wiley-Blackwell, 2005.
Liu, T., Lin, L., Sun, Z., Hu, R., Liu, S., 2010.
J. Biotech. Adv.,
28, 602
e
608.
PROBLEMS
14.1.
Would a cell with a point mutation or a deletion mutation be more likely to revert back
to the original phenotype? Why?
14.2.
You wish to isolate temperature-sensitive mutants (e.g. those able to grow at 25
C but
not at 37
C). Describe experiments to isolate such a cell.
14.3.
An important method for screening for carcinogens is called the
Ames test
.Thetestis
based on the potential for mutant cells of a microorganism to revert to a phenotype
similar to the nonmutant. The rate of reversion increases in the presence of a mutagen.
Many compounds that are mutagens are also carcinogens and vice versa. Describe
how you would set up an experiment and analyze the data to determine if nicotine is
mutagenic.
14.4.
How many different hybridization probes must you make to ensure that at least one
corresponds to a set of four codons encoding the amino acid sequence cal-leu-trp-lys?
14.5.
You wish to develop a genetically engineered
E. coli
producing a peptide hormone. You
know the amino acid sequence of the peptide. Describe the sequence of steps you
would use to obtain a culture expressing the gene as a peptide hormone.
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