Chp 7: Eukaryotic expression:
1. Glycosylation difference: Glycosylation is the addition of specific sugars to certain amino acids to
provide stability and distinctive binding properties to a protein. Proper protein glycoslation is
important because it contributes to protein conformation by influencing protein folding, can target a
protein to a particular location, for example through interaction with a specific receptor molecule; or
can increase protein stability by protecting it from proteases. Oligosaccharides are attached to newly
synthesized proteins in the endoplasmic reticulum and in the Golgi apparatus by specific enzymes
known as glycosylases and glycosyltransferases.
a. “o” glycosylation- attachment to specific sugars to hydroxyl group either serine or threonine.
O-linked oligosaccharides have a number of arrangements with different combinations of
b. “N” glycosylation- attachment of specific sugar to amide group of asparagine. All N-linked
glycosylation in eukaryotes start with the same initial group, which is subsequently trimmed
and then elaborated in diverse ways within and among species.
c. Transfection? Denote inherited changes in animal cells that are due to addition of (forgein
2. General understanding of eukaryotic expression vector?
a. Must have eukaryotic promoter that drives the transcription of the cloned gene of interest,
eukaryotic transcriptional and translational stop signals. Since recombinate DNa is hard to
carry out with eukaryotic cells, most eukaryotic vectors are shuttle vectors with two orgins of
replication and selectable marker genes.
3. Why do you want to use Eukaryotic host organism instead of using E.coli?
a. Fungi share features of other higher eukaryoties and a good choice for heterologous protein
production. They have growth advatnages similar to those of prokyarotes. They generally do
not require growth factors to be added to the growth medium, can correctly process eukaryotic
proteins and secrete large amounts of heterologous proteins.
a. Usefulness of yeast host? Properties of yeast suitable for production of heterologous protein?
i. There are several strong promoters that are isolated from the yeast and characterized,
and a naturally occurring plasmid, called the 2um plasmid, can be used as part of an
endogenous yeast expression vector system
ii. Capable of carrying out many posttranslational modifications
iii. Normally secretes few proteins that when it is engineered for extracellular release of
a recombinant protein, the product can be easily purified.
5. Types of yeast vectors:
a. YEP? (Yeast episomal plasmids) i. based on a high copy number 2um plasmid
b. YIP? (Yeast integrating plasmids)
6. 2 um vector – a small, circular plasmid found in the most natural strains of S. cerevisiase. The vector
replicates independently of the host chromosome via a single origin of replication and is maintained in
more than 30 copies per cell.
7. Can YIP vector replicate inside the host organism? Why or why not?
a. Many S. cerevisiase selection schemes rely on mutant host strains that require a particular
amino acid or nucleotide for growth. Such strains are said to be auxotrophic because minimal
growth medium must be suplmented with a specific nutrient. In practice the vector is
equipped with a functinaonl wild type versone of the gene that complements the mutaed gene
in the host strain.
8. Cu/Zn SOD overproduction-How? And usefulness? Which vector is good?
a. Overproduction of Cu/Zn SOD is used to minimize the cytotoxic effects of superoxide
dismutase SOD. The Cu/Zn SOD scavenges the superoxide radical and combines it with a
hydrogen ion to from hydrogen peroxide which I nturn is degraded to water and oxygen by
catalase or peroxide. In addition, Cu/Zn SOD migh act as a therapeutic agent against
inflammatory diseases. s
9. Pichia Pastoris:
a. Why use this yeast over S. crevasse?
i. Glycoylastion occurs to a lesser extent and the linages between sugar residues are of
a 1,2 type which are not allergenic to humans.
b. AOX 1 gene promoter is used for the expression of…? Transcription of AOX1 is tightly
regulated; in the absence of methanol, the AOX1 gene is completely turned off but responds
rapidly to the addition of methanol to the medium. Therefore, AOX1 promoter is an excellent
candidate for producing large amounts of recombinant protein under controlled conditions.
10. How would you select a yeast strain transformed with a plasmid containing your gene of interest?
Baculovirus: are a large, diverse group of viruses that specifically infect arthopods, including many
insect species and are not infectious to other animals.
Polyhedron- viral nucleocapsids (virons) are clustered in a matrix that is made up of protein
polyhedron. The occluded virions packaged in this protein matrix are referred to as
polyhedron and are protected from inactivation by envirionmental agents.
1. AcMNPV is used for the production of genetically engineered virus? a. AcMNPV is commonly used cell line for genetically enginnered cells. In the cells, the
polyhedron promoter is exceptionally active, and during infections with wild-type
baculovirus, high levels of polyhedron are synthesized.
2. How would you modify this virus (AcMNPV) for the expression of your gene of interest?
a. Create a transfer vector that includes ACMPNV DNA upstream and downstream, used
for homologous recombinant w ACMPNV. Polyhedrin promoter region, MCS,
b. Next, insect cells in culture are cotransfected with ACMNPV DNA and transfer vector
carring the clonged gene.
c. Double-crossover recombination event occurs at homologous polyhedron gene sequences
on the transfer vector and in the ACMNPV genomne and the cloned gene wit the
polyhedron promoter and termination regions becomes integrated into the ACMNPV
3. Why do you linearize the plasmid during cross over?
a. Linearization increase the frequency of recombinant plaques. Two Bsu 361 sites that
were placed on either side of the polyhedron gene. The segment of the essential gene is
meissing so no viral replication occurs. A transfer vector constructed with GOI with
intact versions of gene . the transfer vector is introduced into insect cells that were
previously transfeted with linearized that is missing the segment between the two Bsu
sites. Double crossover event both reestablishes a functional version of ORF and
incorpaotes the coloned gene into the ACMNPV genone
4. What is Bacmid vector?
Mammalian cell expression system:
1. Mammalian vector?
a. Share same features
i. Eukaryotic orgin of replication, usually from a nimal virus such as SV40
ii. Promoter sequences that drive expression of the cloned genes
iii. Selectable marker genes
iv. Transcription termination sequences (polyadenylation signals)
2. Why contain viral promoter?
a. Inducible promoters are often used when continuous synthesis of the heterologous
protein is toxic to the host cell. Expression of a gene of interest is often increased by
placing the sequence for an intron between the promoter and the multiple cloning site
within the transcribed region. 3. What is Kozak sequence?
a. Initiation of translation in higher eukaryotic organism depend on a specific sequence
of nucleotides surrounding the start (AUG) condon in the mRNA. The DNA
sequence for Kozak sequence, which is often followed by a signal sequence to
facilitate secretion, a protein sequence tag to enhance the purification of the
heterologous protein nand a proteolytic leavage sequence that enables the tag to be
removed from the heterologous protein.
4. (fig 7:23 and 7:24)
5. Explain 7:25,
a. Two expression vectors each with the gene or cDNA for one of the subunits and a
different selectable marker gene can be cotransfected into host cells. The transfected
cells are treated with both selecting agents and the cells that surive carry both
vectors. However, two vecotrs are not always maintinaed with the same copy
number so one is overproduced relative to the other and yields of final product
a. single vectors that carry two cloned genes have been developed. Two genes are
placed under the control of independent promoters and polyadenylation signals. To
ensure that equal amounts of recombinant proteins are synthesized, vectors
a. selection schemes are desgined not only to identify transfected cells, but to increase
heterologous protein production by amplifying the copy number of the expression
8. What is IRES and its function? internal ribosomal entry site they allow simultaneous
translation of different proteins from a polycistronic mRNA molecule.
9. What is ‘bicistronic”? to ensure equal amounts of recombinant of recombinant proteins are
synthesized, vectors have been constructed with two cloned genes separated from each other
by a DNA sequence that contains an internal ribosomal entry site.
a. SINGLE TWO GENE TRANSCRIPT SYNTHESIZED AND TRANSALTION
PRODUCEDS from 5’ end of mRNA to produce one of the chains
Molecular Diagnostics: chp 9
1. Generalized ELISA protocol? A way to determine whether an antibody has bound to its target antigen.
Frequently used for diagnostic detection.
a. Bind the sample being tested for the presence of a specific molecule or organism to a solid
support. Wash support to remove unbound molecules b. Add a marker-specific antibody (primary antibody directed against the target antigen) to the bound
material and den wash the support to remove unbound primary antibody. Wash the support to
remove unbound primary antibody
c. Add second antibody that binds to the primary antibody and not to the target molecule.
d. Add the colorless substrate
e. Observe or measure the amount of colored product.
2. Monoclonal vs polyclonal antibody?
a. Create a cell line that could be grown in culture and produce a single type of antibody molecule
with a high affinity for a specific target antigen
b. Such a cell line would provide a consistent and continuous source of identical antibody molecules.
4. Polyclonal antibody
i. The amounts of different antibodies within a polyclonal preparation may vary from one
batch to the next. One batch of polyclonal antibody may have a low level of antibody
molecules directed against a major epitope and not be as effective as a previous antibody
ii. Polyclonal antibodies cannot be used to distinguish between two similar targets
5. Advantages and drawbacks?
6. Method of producing monoclonal antibody?
a. ELISA protocol
7. How would you select hybrid cells? (hybridoma)
8. HAT medium? Why? Function of different chemicals?
a. Growth medium containing hypoxanthine, aminopterin and thymidne
b. Allows only the myeloma spleen fusion cells to grow because none of the other cell types can
proliferate in this medium. Unfused spleen cells and spleen spleen fusion cells cannot grow in this
9. HGRT- myelenoma cells? And their role?
a. The splenic cell suspension is mixed with a suspension of myeloma cells that are genetically
defective for the enzyme hypoxanthine guanine phosphoribosyltransferase