Help for lab report cell biology?

Essentially a lab report for cell biology lab which covers is of dHFR identification with the use of western blotting. I have included all information in the uploaded files and for the full lab report instructions it is after the lab 5 intro where it says full report i have uploaded some images that need to be included aswell. This report is split up into title page, introduction, materials and methods, results, discussion then references.
pcb_3023l_cell_bio_lab_5_analysis_of_recombinant_dhfr.pdf

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pcb_3023l_cell_bio_lab_5_fusion_protein_sequences.docx

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pcb3023l_lab_5_intro_assignment___full_report_instructions.pdf

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Lab 5: Detection and analysis of recombinant dihydrofolate reductase
In this exercise, you will learn about the use and production of genetically engineered
recombinant proteins and will then analyze a series of recombinant fusion proteins by
sequence analysis and Western Blot. For Western Blot, you will be working with both purified
protein and whole cell lysates. The following samples containing human dihydrofolate
reductase (DHFR) protein will be analyzed:
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Purified His-tagged DHFR, expressed and purified from E. coli
Purified GST-tagged DHFR, in vitro-translated from wheat germ extract
Whole cell lysate from HEK293T cells overexpressing Myc-Flag-tagged DHFR
Whole cell lysate control from HEK293T cells
Upon completion of this lab exercise, you should be able to:
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Describe the use of recombinant proteins in research, pharmaceuticals, and other
industries
Identify challenges and considerations for the production of recombinant proteins
Suggest how expression and purification of tagged dihydrofolate reductase proteins
could be used for further research and drug design
Analyze fusion proteins computationally through sequence analysis
Analyze fusion proteins experimentally by Western Blot
Identify potential sources of error/variation based on experimental procedures and
analysis of your results
Background
Recombinant Protein Expression and Purification
The following background material has been adapted from the Protein Expression and
Purification Series, Courtesy of Bio-Rad Laboratories, Inc., © 2011.
Protein Expression and Purification Series
Recombinant Protein Expression and Purification
Why Produce Proteins Recombinantly?
To be used for research, industrial or pharmaceutical purposes, proteins need to be purified in large
quantities. Some proteins, like casein, which makes up 20% of the protein content in milk, can easily be
extracted from a readily available source in large quantities. However, most proteins are not naturally
produced in a form and in amounts that allow easy purification. The techniques of genetic engineering
overcome the limitations of naturally produced proteins by making cells synthesize specific proteins in
amounts which can be purified for use in fundamental research or for industrial and therapeutic
applications.
Biogen, one of the first companies to develop recombinant proteins, is using genetic engineering to
produce human interferon beta-1a in Chinese hamster ovary (CHO) cells and is sold under the
tradename Avonex. A similar form of recombinant human interferon, interferon beta-1b, is expressed in E.
coli and sold by Bayer under the drug name of Betaseron. (An interferon is an immune protein
produced in response to a virus, bacteria, parasite, or tumor cell.) Both recombinant human
interferon beta-1a and 1b have been developed, tested, and brought to the market to help slow
the progression of multiple sclerosis. Without recombinant production of these proteins in CHO cells
or in E. coli, there would not be an easy way to obtain this protein for therapeutic usage.
Table 5.1 Human proteins produced by genetic engineering. Human proteins produced via genetic engineering and the
disease or disorder they are used to treat.
Protein
Used in the treatment of
Insulin
Diabetes
Somatostatin
Growth disorders
Somatrotropin
Growth disorders
Factor VIII
Hemophilia
Factor IX
Christmas disease
Interferon-alpha
Leukemia and other cancers, MS
Interferon-beta
Cancer, AIDS, MS
Interferon-gamma
Cancers, rheumatoid arthritis
Interleukins
Cancers, immune disorders
Granulocyte colony stimulating factor
Cancers
Tumor necrosis factor
Cancers
Epidermal growth factor
Ulcers
Fibroblast growth factor
Ulcers
Erythropoietin
Anemia
Tissue plasminogen activator
Heart attack
Superoxide dismutase
Free radical damage in kidney transplants
Lung surfactant protein
Respiratory distress
alpha 1-antitrypsin
Emphysema
Serum albumin
Used as a plasma supplement
Relaxin
Used to aid childbirth
Protein Expression and Purification Series
Choice of Cell Type
The biotechnology industry uses several cell types, both prokaryotic (bacteria) and eukaryotic
(animal, fungi, plant), to synthesize recombinant proteins. The choice of the host cell depends on
the protein expressed.
Bacteria can express large amounts of recombinant protein, but the expressed proteins
sometimes do not fold properly. In addition, bacterial cells cannot carry out the post-translation
modifications that are characteristic of some of the proteins made by eukaryotic cells. The most
important post-translation modification is glycosylation, the covalent addition of sugar residues to
the amino acid residues making up the protein. Glycosylation can change the structure and thus
affect the activity of a protein. Many mammalian blood proteins are glycosylated, and the addition
of these sugars often changes the rate of turnover (half-life) of the protein in the blood, because
proteins that are misfolded will be quickly degraded. If glycosylation is important for the function
of the protein, mammalian cells are the cell type of choice, but these cells produce less protein
and are more expensive to grow.
In the early days of the biotechnology industry Escherichia coli (E. coli) was the bacterial host of
choice. This species had been used as the primary experimental system to study bacterial
genetics for decades. More was known about the molecular biology of E. coli than any other
species, and many genetic variants were available. In addition E. coli grows quickly, can reach high
cell concentrations, and can produce large quantities of a single protein. It is also relatively
inexpensive to grow. Today E. coli remains the bacterial system of choice, and many companies
produce recombinant proteins using this bacterial species. Insulin, the first protein produced by
genetic engineering, was produced in E. coli. Blockbuster products like human growth hormone and
granulocyte colony stimulating factor (which increases white cell production in cancer
chemotherapy patients) are also produced using this bacterial species. In general, if a protein’s
properties allow it to be produced in bacteria, then E. coli is the system of choice.
For recombinant protein expression in lower eukaryotic cells, two yeast species are commonly used:
Saccharomyces cerevisiae (S. cerevisiae) and Pichia pastoris (P. pastoris). S. cerevisiae is the yeast species
used to make bread, wine and beer. Baker’s yeast is used in research laboratories as a model
system to study the genetics of eukaryotic cells. P. pastoris is a yeast species initially discovered by
the petroleum industry. It divides rapidly, grows to a very high cell density, and can produce large
quantities of a single protein. In addition, it can be genetically engineered to secrete the protein
into the surrounding medium to allow easier recovery. Both species can glycosylate proteins,
although the glycosylation patterns may differ from mammalian patterns. The sugars that are
added to the protein and their position on the amino acid chain may differ between yeast and
mammalian cells. Despite these advantages, relatively few biotech companies use yeast as a
production system. The exceptions are the vaccine that immunizes against hepatitis B virus and
the vaccine Guardasil that immunizes against HPV, the human papillomavirus.
If a protein has a very large and complex structure, or if that protein requires glycosylation to be
active, then the protein must be produced in a mammalian cell line. Chinese hamster ovary cells
(CHO) is the cell line that is almost always used. CHO cells bear relatively little resemblance to the
cells of the hamster from which they were derived in the 1950s; they have adapted to growth in
cell culture medium. Cell lines are established when cells from a multicellular organism are
separated from one another by a protein-digesting enzyme and grown as if they are really a
unicellular organism. The cells require a rich medium that provides them with all of the amino
acids, vitamins, and growth factors that they need to grow. This complexity means that
mammalian growth medium is many times more expensive than the media used to grow either
bacterial or yeast cells. The CHO cell lines can be adapted for growth in suspension culture. The
CHO cell lines are most often engineered to synthesize the protein of interest on the ribosomes
Protein Expression and Purification Series
attached to the rough endoplasmic reticulum, to package and glycosylate the protein in the
Golgi apparatus, and to eventually secrete the protein into the extracellular medium where it is easier
to purify.
CHO cells can glycosylate proteins with a mammalian glycosylation pattern. If glycosylation is
important to the function of the protein, CHO cells should be used. Because CHO cells divide
slowly, the production runs are much longer than with E. coli (on the order of weeks rather than
days). All equipment and all growth media must be scrupulously sterilized. A single contaminating
bacterial cell will overgrow the culture and will lead to that batch being discarded. Although the
growth of CHO cells takes longer, uses expensive media, and presents a greater risk of
contamination, the isolation of the proteins that these cells produce is usually easier than in
bacterial or yeast cells.
Interferon beta provides a good example of how the end product influences the choice of
expression system for recombinant proteins. Avonex, the human interferon beta-1a form
produced in CHO cells, is glycosylated; while Betaseron, the human interferon beta-1b form
produced in E. coli, is not glycosylated. Since glycosylation is important for interferon beta-1a
function, it is produced in CHO cells.
Table 5.2 Advantages and disadvantages of using bacteria, yeast and mammalian cells to produce recombinant
proteins.
Parameter
Bacteria
Yeast
Contamination risk
Low
Low
Mammalia
n
High
Cost of growth medium
Low
Low
High
Product titer (concentration)
High
High
Low
Folding
Sometimes
Probably
Yes
Glycosylation
No
Yes, but different pattern
Full
Relative ease to grow
Easy
Easy
Difficult
Relative ease of recovery
Difficult
Easy
Easy
Deposition of product
Intracellular
Intracellular or extracellular
Extracellular
Product
Intracellular
Often secreted into media
Secreted
Table 5.3 Examples of pharmaceutical products and the cell line used to produce them.
Product
Cell Line
Insulin
Escherichia coli
Human growth hormone
Escherichia coli
Granulocyte colony stimulating factor
Escherichia coli
Tissue plaminogen activator
CHO cells
Pulmozyme (DNase) cystic fibrosis
CHO cells
Erythropoietin induces red blood cell production
CHO cells
Hepatitus B virus vaccine
Yeast
Human papillomavirus vaccine
Yeast
Rituxan rheumatoid arthritis, non-hodgkins lymphoma, leukemia
CHO cells
Herceptin breast cancer
CHO cells
Choice of Plasmid
Once the cell type has been chosen, the plasmid or vector to express the protein needs to be
selected. Different plasmids are used for expression of proteins in bacteria, yeast and higher
Protein Expression and Purification Series
eukaryotic cells. Some features that need to be considered in the plasmid include: selection
system (such as antibiotic resistance), the promoter, the copy number of the plasmid,
presence of signal peptide sequence to excrete the expressed protein out of the cell,
presence of sequence coding for a protein purification tag or DNA coding for fusion protein
partners.
Antibiotic resistance is a common component of both prokaryotic and eukaryotic vector systems. The
presence of a gene for antibiotic resistance allows for selective retention of the plasmid and
suppression of growth of any cells that do not contain the plasmid. However, to ensure safety
for expression systems being used for vaccine and therapeutic protein production, other
selection systems can be used such as the expression of a required metabolic enzyme that has
otherwise been deleted from the host organism.
The promoter controls the level of gene expression. It can be either constitutive, meaning that it is
always active and there is no control over when the protein of interest is expressed, or
inducible, meaning that its activity can be triggered by external factors. Examples of inducible
promoters are the heat shock promoters, which are activated by a change in temperature.
These promoters are derived from naturally occurring sequences in organisms that need to
express a different protein when they are in a warm environment versus a cold one. Other
inducible promoters are activated by the addition of a chemical such as lactose or its analog IPTG
in the case for the LacZ promoter in E. coli. The T7 promoter is an example of a chemically induced
promoter system. For industrial applications, genes for the protein of interest tend to be under
inducible control.
The copy number of a plasmid depends
on the origin of replication present in the
plasmid. The origin of replication
determines the level of control of replication
of the plasmid, and if the plasmid is under a
relaxed control more copies can be made.
The size of the plasmid and size of the
insert also affect the number of copies. If
there are more copies of the plasmid in cells
it is possible for them to produce more
protein than cells that have fewer copies of
the plasmid. Plasmids used for cloning
such as pUC tend to be higher copy
number while plasmids used for protein
expression tend to be larger and have
lower copy number.
Prokaryotic Cells
Eukaryotic Cells
Bacterial/Yeast Culture
Cell Culture
Cell Lysis and Protein Extraction
To facilitate the purification of the
expressed protein, DNA sequences coding
for a signal (amino acid sequence) that
targets the protein of interest to be
secreted into the periplasmic region of
E. coli or into the extracellular medium for
eukaryotic cells, can be fused to the
gene of interest. Other tags that are
commonly added are fusion proteins to
increase the solubility of the expressed
protein (such as glutathione-SFigure 5.1 Gene Design For Recombinant Protein Production.
Protein Expression and Purification Series
transferase, GST) or affinity tags (polyhistidine or GST tags) that can be used to selectively purify
the recombinant protein.
Choice of Cell Expression System
Once the cell expression system has been determined for recombinant protein production, the
gene construct to express the recombinant protein needs to be designed. This could be as
simple as taking the coding sequence of the gene of interest as it exists in the parent organism
and inserting this sequence into an expression plasmid for the cell system being used. However,
this usually does not produce optimal levels of recombinant protein when the gene is expressed
in a heterologous system (a cell or organism different from the one where the gene is naturally
found) because the preferred codon usage for various species differs. For example, the codon
GGA for glycine can be found at a frequency of 16.4 times per 1000 codons in human genes
while it is only used 9.5 times per 1000 codons in E. coli genes. Therefore, there is a chance that
leaving this codon in the recombinant gene might lead to lower levels of recombinant expression
due to a scarcity of tRNA molecules for GGA.
A second consideration for recombinant gene design is whether or not the protein of interest is expressed in a
soluble or in an insoluble form. If a protein is expressed in an insoluble form, it can be easier to initially
separate it from components, such as nucleic acids, phospholipids and soluble proteins, by
centrifugation. An insoluble protein is also relatively protected from the action of proteolytic proteins
that are present in the host cell that can be released upon lysis of the cell. However, if a fully
functional recombinant protein is desired, it is necessary to refold the insoluble protein to its native
conformation, which many times proves extremely problematic especially if the fully refolded protein
has disulfide bonds and multiple subunits.
The ability to express a recombinant protein in the soluble form is partially dependent on the protein
being expressed as well as the rate of expression of the recombinant protein. If the protein is
expressed at an extremely high rate, it could overwhelm the native proteins involved in folding
proteins (such as chaperonins) in the cell host. The rate of expression can be controlled by the
promoter system involved such as the T7 polymerase system used commonly in E. coli.
A final consideration for recombinant protein gene design is how the recombinant protein will be
purified from the other host cell components. Some recombinant proteins, such as antibodies,
have a specific antigen against which they were raised and hence can be purified by binding to
that molecule (affinity chromatography).* Other recombinant proteins have a very large positive
or negative charge associated with them and can be purified by binding to charged resins (ion
exchange chromatography). Some proteins do not have any strong distinguishing property, and
an affinity tag, such as GST or a histidine tag can be added as a DNA sequence to the gene of
interest at either the 5′ or 3′ end of the recombinant gene. The tag attached to the protein
enables the specific purification of the recombinant protein using affinity chromatography
methods.
*Antibodies can also be purified by binding to Protein A, a surface protein of Staphylococcus aureus which has
high affinity for immunoglobulins. Protein A is commonly used for the first step in the purification of antibodies
in industrial applications.
DHFR—Our Protein of Interest
The focus in the experiment here is on the protein dihydrofolate reductase (DHFR), which is
essential for proper cell function and illustrates the importance of basic oxidation– reduction
enzymatic reactions. DHFR is an enzyme that converts
Protein Expression and Purification Series
dihydrofolate, a folic acid derivative, into tetrahydrofolate (THF) by
the addition of a hydride from NADPH. Tetrahydrofolate is a
methyl group shuttle required for the synthesis of purines,
thymidylic acid, and amino acids, all essential for nucleic acids.
DHFR is ubiquitous in prokaryotic and eukaryotic cells, and is found
on chromosome 5 in humans. Deficiency in DHFR has been linked
to megaloblastic anemia, an anemia disorder with larger-thannormal red blood cells, as well as cerebral folate metabolism
disorders. Both are treatable with folic acid and/or Vitamin B12,
depending on symptoms. Being able to control DHFR makes it a
powerful tool not only for research and gene manipulation but
also for medical treatments for cancer and malaria. When DHFR
is inhibited or reduced, it leads to a shortage of thymidylates,
interfering with nucleic acid synthesis. A lack of nucleic acid
synthesis thus interferes with cell growth, proliferation, and
ultimately causes cell death.
DHFR Cancer Connection
Cancer occurs when a particular cell loses the ability to control its
division. These dividing cells spread, displace normal cells, disrupt
the architecture of tissues, and use up the nutrients required by
normal cells. Surgery can remove most of the cells in a solid
tumor, but malignant cancers send out colonizing cells called
metastases that use the blood and lymph systems to spread far from
the tumor and non-solid tumors, such as leukemia or lymphoma,
are not confined to one specific area. Clinicians use radiation or
chemotherapy to kill these cells.
Figure 5.2 DHFR protein structure.
Chemotherapy drugs target cancer cells by disrupting the functions of actively dividing cells. This
strategy exploits the fact that most of the cells in an adult are not dividing. Therefore, chemotherapy
damages the rapidly dividing cancer cells by disrupting structures required for mitosis, like spindle
fibers, o …
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