Chapter 6 and 7 Answers
Chapter 6:
1. There are a number of ways to ensure maximal expression of a cloned gene. Firstly, you
must select the proper vector for your target gene. This includes thinking about which
promotor sequence to use, which terminator sequence to use, what strength of ribosome
binding site will be needed. For example, the use of a promoter that in the host organism
would be constitutively active. In addition considerations can be taken about what host
system to use, including the approximate number of copies of gene, efficiency of
translation, final location of protein product, and the resting location of gene.
3. The promoter that stimulates the most expression may not be the best selection because
this can often be too much of an energy drain for the host organism. The production of
protein (transcription and translation) takes energy so producing large quantities
consumes large amounts of energy. In addition, if your gene product is harmful to the host
organism producing large amounts may kill the cell.
6. A fusion protein is useful for a couple of different reasons. Firstly, fusing your target
protein to another protein can often prevent your target protein from being degraded by
proteases present in the cell. In addition, the extra piece that you add to the target protein
can be designed to allow for selection of your protein. For example a histidine tag could be
added to the target protein because repetitive histidine sequences are rare so one can use a
column with Ni2+ to separate the fused protein from other proteins to purify their product.
The added part usually contains a restriction enzyme domain so that it can easily be
removed once the fusion protein has been isolated.
8. It is sometimes advantageous to express a protein on the surface of a bacterium or
bacteriophage because they can be easily identified by immunological assay with
antibodies. This technique also allow for high expression of the product. This is partially
because since the protein is exterior to the cell you do not have to worry about degradation
from internal proteases.
20. It is possible to engineer a gene so that the protein product will be secreted to the
medium by E. coli cells. This process is easiest if you select a gram positive bacterium
because they lack an outer membrane. However, it is still possible to secrete a protein
from a gram negative host such as E. coli via molecular manipulation. A bacteriocin release
protein activates phospholipase A, a component of the inner membrane. Phospholipase A
cleaves phospholipids in both the inner and outer membrane. This allows some internal
proteins to escape into the medium. To do this one must use E. coli cells that already
contain the bacteriocin releasing protein gene.
Chapter 7:
1. Endoplasmic Reticulum:
The major posttranslational modifications in the ER are the folding assistance by the
chaperones BiP and calnexin as well as the formation of disulfide linkages between
cysteine residues with protein disulfide isomerases. In addition some glycoslyation occurs
in the ER.
Golgi Apparatus:
The major posttranslational modification that occurs in the Golgi is protein
glycoslyation. Specifically, the addition of a carbohydrate on the hydroxyl groups of serines
or threonines or on the amide groups of asparagine.
Other modifications include phosphorylation, acetylation, sulfation, acylation, gammacarboxylation, myristylation, and palmitoylation.
2. N linked glycosylation patterns differ slightly between yeast, insects, and mammals. In
most cases the differences has to do with how elaborate the modifications are. The initial
core group of sugars that are added seem to be mostly conserved across eukaryotes but
after that the glycosylations become more variable.
3. A eukaryotic expression vector must have a eukaryotic promoter that will drive
transcription, eukaryotic transcriptional and translational stop signals, a sequence that will
facilitate polyadenylation of the mRNA product, and a selectable gene marker. Design must
be planned so that each sequence that must be incorporated into the vector is
complementary and will insert so that the sequence can be functionally transcribed.
7. Baculoviruses are viruses that specifically infect insects. The original baculovirus
expression system is designed for the baculovirus AcMNPV. A segment of the AcMNPV
DNA is inserted into a plasmid vector with the MCS site interior and the plasmid is
proliferated in E. coli. Next, the insect cells are exposed to the AcMNPV DNA and the
transfer vector that carries the cloned gene. In some of the cells double-crossover
recombination occurs and the plasmid picks up the AcMNPV DNA and loses the polyhedrin
gene. These cells lyse and recombinant baculovirus can be isolated. These baculoviruses
contain the target gene. This technique has been improved via linearization of the AcMNPV
DNA before transfection, which increases the frequency of recombinant plaques. In
addition Bsu36I sites have been added on both sides of the polyhedrin gene which facilitate
the integration of the target gene into the AcMNPV genome. Over 90% of the plaques are
recombinant with these improvements.
10. There are multiple ways to select for expression of your gene. Depending on the
identity of your protein there may be an observable change that occurs that will confirm
that your gene is being expressed. However, this will not always be the case. Geneticin or
G-418 is commonly used as a selectable agent in eukaryotes. The plasmid carries a gene
(neo) encoding neomycin phosphotransferase that blocks geneticin’s action of breaking
DNA strands and preventing protein synthesis. Therefore, only cells that have been
transformed survive after geneticin treatment. Another example of a selctable agent is
puromycin. This agent inhibits protein synthesis in cells that have not be transformed with
a vector containing the Pac gene encoding Puromycin-N-acetyltransferase.
11. A stable mammalian cell line differs from a transient one in that transient lines are
used for rapid production of small amounts of protein so that one can simply determine if a
protein is good for something such as a drug treatment. These cells can grow in serum free
medium and grow to high density relatively quickly. In contrast cells used for stable gene
expression are used when large amounts of protein product are needed.
13. Chromatin is the term for the DNA and associated proteins such as histones that are
compacted together to form chromosomes. It is important your gene of interest is
incorporated into euchromatin, which is more loosely packed, so that it will actually be
expressed. Compact heterochromatin is not accessible and if your target gene is
incorporated in this chromatin it will not be highly expressed. There are a number of
techniques designed to accomplish this such as modifying host strains so that more histone
acetyltransferases are produced so that more chromatin will be in euchromatin form. This
increased expression should be targeted to the area where you want to insert your gene to
ensure maximum expression. It is also useful to incorporate a DNA segment known as a
STAR element which blocks deacetlyation and methylation to keep the DNA accesible.
Chromatin opening sequences which are found near promoters can also be added to the
target gene sequence to loosen up the chromatin.
14. There are a number of criteria that go into deciding what eukaryotic expression
system, insect, yeast, or mammalian, should be used. Mammalian systems are used when a
protein product requires full complements of posttranslational modification. This system
is challenging because mammalian cells are easily stressed and many will die. Genetic
modifications are used to keep them alive and healthy. Yeast systems are used when the
protein product would not require much posttranslational modification. This system is
used because the cells are easier to maintain and grow more rapidly. However, yeasts
often lack many of the proteins required for proper folding of the target protein so they
may not produce functional product. Insect systems are used for proteins that are
nonfunctionally produced in yeasts because insect posttranslational modifications are
more similar to that of mammals. This system can more rapidly produce product than the
mammalian system and the cells are easier to maintain. In summary, less expensive
expression systems are favored unless authenticity of an important recombinant protein
can be obtained only with mammalian cells (which are the most expensive to grow).