Transcript proteinch10_2015-05-27.ppt

Recombinant Proteins
Proteins and Recombinant DNA TechnoIogy
Expression of Eukaryotic Proteins in Bacteria
Translation Expression Vectors
Codon Usage Effects, ,
Avoiding Toxic Effects of Protein Overproduction
Increasing Protein Stability
Improving Protein Secretion
Protein Fusion Expression Vectors .
Expression of Proteins by Eukaryotic Cells
Expression of Proteins by Yeast
Expression of Proteins by Insect Cells
Protein glycosylation
Expression of Proteins by Mammalian Cells
Expression of Multiple Subunits in Mammalian Cells
Comparing Expression Systems
Why express proteins?
Expression of Eukaryotic Gene
???????
THE RANGE OF INDUSTRIALLY SIGNIFICANT PROTEINS
A wide variety of proteins find industrial application.
These include
enzymes,
antibodies,
hormones,
blood factors,
growth factors and
regulatory factors.
Such proteins are employed as
therapeutic and
diagnostic agents and
in the manufacture of a wide variety of biologically derived
industrial commodities.
Biofuels
Green
energy
2011 the global market valued US$3.5 billion
2011
Novozymes 47%
DuPont (DuPont acquired Genencor) 21%
Technical enzymes are valued at just over $1 billion in 2010. This sector will increase at
a 6.6% CAGR (compound annual growth rate ) to reach $1.5 billion in 2015.
The highest sales: leather market bioethanol market.
The food and beverage enzymes: $1.3 billion by 2015, $975 million in 2010
CAGR of 5.1%.
dairy market: $401.8 million in 2009.
PROTEINS AND RECOMBINANT DNA TECHNOLOGY
(血友病
硬化症;
肉芽腫
2015: US$ 143.4 Billion
monoclonal antibodies in the advanced clinical phase, may enter the market.
Largest Market of monoclonal antibodies(46.4%) followed by Insulin, Interferon,
Erythropoietin, Blood clotting factors, Granulocyte colony-stimulating factors,
Human growth hormone and Follicle stimulating-hormones respectively.
Roche,
Amgen,
Merck & Co.,
Eli Lilly,
NovoNordisk,
Johnson & Johnson
SUBMITTED BY:
Aditya Kanwal- 2012BT09Kishan R. Bharadwaj- 2012BT11M.Tech
BiotechnologyMNNIT
Adalimumab binds to tumor necrosis factor-alpha (TNFα).
It fuses the TNF receptor to the constant end of the
IgG1 antibody.
is an immunomodulator drug currently used to treat multiple sclerosis.
is a chimeric monoclonal antibody against the protein CD20, which is primarily found on t
VEGF antagonist
It consists of microcrystals that slowly release insulin, giving a long duration of action of 18
profile (according to the insulin glargine package insert).
Parenteral administration of biopharmaceuticals
Injection, infusion, and implantation
Virtually, all therapeutic peptides and proteins are delivered parenterally.
Gene immune responses safety
Industrial interests 100-1000 NT/mg
Interferon- (E. coli) 120,000
insulin (synthetic)
Tissue plasminogen activator 24,000
erythropoietin
Monoclonal antibodies
100,000
2200
100,000
9.2 Requirements for protein production from cloned genes
Heterologous expression ???
Requirements: 1.
2.
3.
4.
gene
The
for the protein
has to be placed downstream from a strong promoter, to maximize the amount of
mRNA which is produced for translation into protein.
vectors
Ideally, there will be many copies of this gene present in each cell, which will
increase the amounts of mRNA and hence of protein which are produced. This is
done by placing the gene and its promoter into a suitable vector, typically a
plasmid.
The protein needs to be produced in such a way that it does not harm the host,
at least until high levels of the protein have accumulated.
Finally, the protein ideally needs to be in a soluble, active form which can be
easily purified away from the other components of the organism, such as other
proteins, lipids, metabolites and nucleic acids, all of which might interfere with the
uses to which the protein is going to be put.
cDNA (open-reading frame), vectors, expression host (E. coli, yeast, ….), methodology (transformation, purification….)
9.2 Requirements for protein production from cloned genes
Heterologous expression ???
Requirements:
1. genes
2. vectors
3. hosts ???? transformation
4. Systems for identification and purification ????
cDNA (open-reading frame), vectors, expression host (E. coli, yeast, ….), methodology (transformation, purification….)
Expression of Eukaryotic Gene in Bacteria
Expression of Eukaryotic Gene
???????
E. coli ori
Cloning host
Expression host
E. coli selection marker
Host selection marker
FIGURE 10.1
Expression of Eukaryotic Gene in Bacteria—Overview Eukaryotic genes must be adapted for
expression in bacteria. First, the mRNA from the gene of interest is converted to cDNA to
provide uninterrupted coding DNA. The cDNA is cloned between a bacterial promoter and a
bacterial terminator so the bacterial transcription and translation machinery express the
coding sequence.
Even if a cloned gene is transcrbed at a high level, production of the encoded protein may
be limited at the stage of protein synthesis. Different mRNA molecules are translated with
differing efficiencies. Several factors are involved:
(a) Strength of the ribosome binding site ribosome interaction
[b] mRNA stability and/or secondary structure
(c) Codon usage
Protein sources
• Microorganisms as sources of proteins.
• Protein production by genetically engineered microorganisms.
Heterologous protein production in E. coli.
Advantages
Genetic characteristics of E. coli are well established
Suitable fermentation technology is well established
Can generate potentially unlimited supplies of the recombinant protein
Economically attractive
Disadvantages
Intracellular accumulation of recombinant protein in inclusion bodies
Unable to undertake post-translational modifications of protein
Adverse public perception of products produced by recombinant
methodologies
post-translational modification : glycosylation S-S
N-formylmethionine
lipopolysaccharide endotoxin
Vectors for gene expression
E. coli
Maximizing the expression of cloned genes in E. coli
Factors affecting the expression of cloned genes
Promoter strength
Translational initiation sequences
Codon choice
Secondary structure of mRNA
Transcriptional termination
Plasmid copy number
Plasmid stability
Host cell physiology
A. Constructing the optimal promoter
constitutive promoters
drawback???????
regulatory (inducible) promoters
The two most important features of a promoter are its strength
and the degree to which expression from it can be regulated
The first is important in achieving high levels of expression: in general, the stronger the
promoter, the more mRNA will be synthesized, and the more protein will result from the
translation of this mRNA.
TRANSLATION EXPRESSION VECTORS
Bacterial ribosomes bind mRNA by recognizing the ribosome
binding site
(RBS) (also known as the Shine-Dalgarno sequence).
The RBS base pairs with promoter and the sequence AUUCCUCC on the 16S rRNA of
the small subunit of the ribosome.
Expression vectors are designed to optimize gene expression at the level of transcription.
However, it is also possible to design translational expression vectors to maximize
the initiation of translation.
These vectors possess a consensus RBS plus an ATG start codon located an optimum
distance (8 bp) downstream of the RBS.
FIGURE 10.2
Translational Expression Vector The recognition site for NcoI is C/CATGG, which has an ATG in
the middle. The gene of interest and the translation vector each have an NcoI site, allowing the gene
of interest to be cloned exactly into the correct site for highest protein expression. The vector also has
a consensus RBS spaced 8 base pairs from the ATG, which provides an optimum binding site for
ribosomes. The terminator sequence(s) is also strong to prevent transcriptional run-on.
The secondary structure
effects on the level of translation.
of the mRNA may have significant
If the mRNA folds up so that the RBS and/or start codon are blocked, then
translation will be hindered. In particular, the sequence of the first few codons of the
coding sequence should be checked for possible base pairing with the region around the
RBS.
If necessary, bases in the third (redundant) position of each codon may be changed to
eliminate such base pairing. Getting active and abundant translation is the key to
preventing mRNA instability. Any mRNA that is not being actively translated becomes
subject to degradation, which decreases the protein yield.
CODON USAGE EFFECTS
When genes from one organism are expressed in different host cells the problem of
codon usage arises.
For example, the amino acid lysine is encoded by AAA
or AAG. In E. coli,
AAA is used 75% of the time and AAG only 25%.
In contrast, Rhodobacter does the exact opposite and uses AAG 75% of the time, even
though E. coli and Rhodobacter are both gram-negative bacteria.
When a cell uses a particular codon only rarely, it has lower levels of the tRNA that
reads the rare codon. Genes that have been codon-optimized for new host
organisms may show a 10-fold increase in the level of protein produced, due to more
rapid elongation of the polypeptide chain by the ribosome.
Over-express genes of rare tRNAs in E. coli
FIGURE 10.3
Codon Usage Affects Rate of Translation Bacteria prefer one codon for a particular amino acid to
other redundant codons. In this example, the ribosome is stalled because it is waiting for lysine tRNA
with a UUU anticodon. Escherichia coli does not use this codon very often and there is a limited
supply of this tRNA.
AVOIDING TOXIC EFFECTS OF
PROTERN OVERPRODUCTlON
Although higher yields are usually desirable, overproduction of a recombinant protein
may harm the host cell. In bacteria, when too much protein is manufactured too fast,
the surplus forms inclusion bodies. These are dense crystals of misfolded and
nonfunctional protein (see below).
Thus expression systems for recombinant proteins have features to control when and
how much protein the host cell makes.
Two common expression systems are
the pET and pBAD
systems for E. coli.
These have control mechanisms to switch recombinant protein production on or off and
the pBAD system can also modulate the amount of protein produced.
The simplest examples of promoters for protein expression are those
which are derived from operons of E. coli, with
the lac promoter of
the lac operon,
You may already know that expression from the lac promoter is regulated by the lac
repressor protein, LacI
or isopropyl--D-thiogalactopyranoside
(IPTG)
CRP protein: a positive
activator that binds in the
absence of glucose.
In the absence of lactose in the growth medium, the E. coli lac promoter is repressed,
i.e., turned off, by the lac repressor protein, which prevents the lac operon from being
transcribed.
Induction, or turning on, of the lac promoter is achieved by the addition of either lactose
or isopropyl--D-thiogalactopyranoside (IPTG) to the medium. Either of these
substances prevents the lac repressor from binding to the lac operator, thereby
enabling transcription to occur.
Although it is widely used, the lac promoter is not always the best choice
for expression of a heterologous protein in E. coli.
First, it is weaker than some other promoters, and so maximal levels of protein
expression will not be achievable if it is used.
Second, it is rather "leaky", which means simply
that even under conditions where no IPTG or lactose is present in the
medium, a fair amount of transcription can still occur from this promoter.
T7 RNA polymerase
E. Coli promoter
FIGURE 10.4
pET Protein Expression System(A) Recombinant proteins are not expressed in the cell until induced.
The pET plasmid has the gene for LacI protein, which represses both the gene for T7 RNA
polymerase on the bacterial chromosome and the recombinant protein gene on the pET plasmid. (B)
When the inducer IPTG is added, it causes release of LacI from both promoters. The gene for T7 RNA
polymerase is then expressed. This polymerase then transcribes the gene for the recombinant protein.
The effectiveness of deactivating a repressor protein and thereby activating transcription
depends on the ratio of the number of repressor protein molecules to the number
of copies of the promoter sequences.
leaky
In systems that use the lac promoter, a mutant form of the lacI gene (lacIq) produces
much higher levels of the lac repressor, thereby decreasing the leakiness under
noninduced conditions, i.e., transcription of a cloned gene in the absence of inducer.
The pBAD system is based on the arabinose operon.
This is induced by adding arabinose, which binds to the AraC regulatory protein.
Activated AraC exits the 02 site and binds to the I site (Fig. 10.5). This activates
transcription of the cloned gene. The pBAD system is modulated by the amount of
arabinose added to the culture. If a lot of recombinant protein is needed, then more
arabinose is added. However, if the recombinant protein is toxic to the host cells,
then less arabinose is added, and less recombinant protein is made. This effect is
based on the culture, not the cell. Each cell manufactures the recombinant protein in
an "all-or-none‘ fashion. Low levels of arabinose will not activate every cell in the
culture, whereas higher levels of arabinose do activate every cell.
Regulation of the araBAD operon by the
combined action of CAP and AraC Protein
Figure 29.17
FIGURE 10.5
pBAD Expression System(A) Recombinant proteins are not expressed when the AraC protein dimer
binds to the O1 and O2 regulatory regions on the pBAD plasmid. (B) When arabinose is present, the
sugar induces AraC to switch conformation, and it now binds to the O1 and I sites. This conformation
stimulates transcription of the recombinant protein.
INCREASING PROTEIN STABILITY
Different proteins vary greatly in stability. The lifetime of a protein inside a cell depends
mainly on how fast the protein is degraded by proteolytic enzymes or proteases.
(a) The identity of the N-terminal amino add greatly affects the half-life of proteins.
Although all polypeptide chains are made with methionine as the initial amino add,
many proteins are processed later. Therefore the N-terminal amino add of mature
proteins varies greatly. The
N-terminal rule for stability is shown in Table 10.2.
(b] The presence of certain internal sequences greatly destabilizes proteins. PEST
sequences are regions of 10 to 60 amino adds that are rich in P
(proline), E
(glutamate), S (serine), and T (threonine). The PEST sequences create
domains whose structures are recognized by proteolytic enzymes.
One proper folding method to alleviate the aggregation is to
provide
molecular chaperones that help fold the recombinant protein
correctly. Molecular chaperones attach themselves to polypeptides while they are
being translated and help keep the protein molecular unfolded until translation is
complete. Then the protein can fold into its correct shape (Fig. 10.6).
IMPROVlNG PROTEIN SECRETION
When a bacterial cell synthesizes a recombinant protein, it may end up in the
the periplasmic space
cytoplasm,
between the inner and outer
membranes, or be exported out of the cell into the culture medium.
Secretion across the inner, cytoplasmic, membrane is directed by the presence of a
hydrophobic signal sequence at the N-terminal end of the newly synthesized protein.
The signal sequence is cut off after export by signal peptidase (also known as
leader peptidase).
Although bacteria such as E. coli export few proteins, special secretion systems do exist
that allow export of proteins across both membranes into the culture medium.
It is obviously easier purify a secreted protein than one that remains in the cytoplasm
with the majority of the bacterial cell's own proteins.
Several approaches exist for this:
a) A signal sequence is engineered into the cloned gene.
General secretory system efficieency
b) The
recombinant protein may be fused to a bacterial protein
that is normally exported .
The maltose binding protein of E. coli is efficiently exported to the periplasmic space
and is a favorite carrier protein for recombinant protein.
C) The
gene of interest can be express in gram-positive
bacteria, such as Bacillus, which lack an outer membrane. Yeast animal cells
d) Secretion
across both membrane of gram-negative bacteria
such as E. coli may be achieved by specialized export
system.
Type secretory system
hemolysin
FIGURE 10.7
Secretion across Both Membranes. Protein secretion in E. coli involves a general secretory system
that recognizes a signal sequence and exports those particular proteins. The protein is only
transported to the periplasmic space. Type I secretory systems transport the protein from the
cytoplasm, through the periplasmic space, to the outside of the cell. Type II secretory systems take
proteins already in the periplasmic space and transport them across the outer membrane.
PROTEIN FUSION EXPRESSION VECTORS
Joining the coding sequences of two proteins together in frame makes a protein fusion
(see Chapter 9). Consequently, a single, longer polypeptide is made during translation. If
the first (i.e., N-terminal) protein is normally secreted, then the fusion protein will be
secreted, too.
Thus it is possible to achieve export of a recombinant protein by joining it to a protein
that the cell normally exports.
Protein fusions also help address the issues of
stability and
purification. Many eukaryotic proteins are unstable inside the bacterial cell.
This is especially true of growth factors, hormones, and regulatory peptides, which are
often too short to fold into stable 3D configurations.
Solubility and cellular localization
A vector can enhance solubility and/or folding in one
of three ways:
1) provide for fusion to a polypeptide that itself is highly soluble [e.g.
glutathione-S-transferase (GST),
thioredoxin (Trx),
N utilization substance A (NusA)],
2) Provide for fusion to an enzyme that catalyzes disulfide bond formation (e.g.
thioredoxin, DsbA, DsbC),
3) provide
a signal sequence for translocation into the
periplasmic space.
When using vectors designed for cytoplasmic expression, folding can be improved in
hosts that are permissive for the formation of disulfide bonds in the cytoplasm (e.g.
trxB and gor mutations).
Fusion Proteins
Cleavage of Fusion Proteins
One method which is often used to deal with
this problem is to incorporate codons for a sequence of amino acids
between the protein of interest and the protein or peptide that it is fused to,
the sequence being recognized by a protease which can cleave the peptide
bond. The most widely used protease is factor Xa, a protease whose normal
role is in the blood coagulation cascade. Factor Xa recognizes the amino
acid sequence Ile-Glu-Gly-Arg and cleaves the peptide bond which is on
the C-terminal side of the arginine residue.
EXPRESSION OF PROTEINS BY EUKARYOTIC
CELLS
A variety of eukaryotic modifications may occur after the polypeptide chain has been
made (Fig. 10.8).
These include:
[a] Chemical
chain.
modifications that form novel amino acids in the polypeptide
[b) Formation of disulfide bonds between correct cysteine partners (e.g.,
the assembly of insulin, Chapter 19).
(c] Glycosylation, that is, the addition of sugar residues at specific locations on the
protein. Many cell surface proteins are glycosylated and will not assemble correctly
into membranes or function properly if ladring their glycosyl components.
[d] Addition of a variety of extra groups, such as fatty add chains,
acetyl groups, .phosphate groups, sulfate groups.
(e) Cleavage of precusor proteins. This may occur in several stages, as
illustrated by insulin (see Chapter 19). Cleavage may be involved with secretion, correct
folding, and/or activation of proteins.
FIGURE 10.8
Protein Modifications in Eukaryotes(A) Novel amino acids may be inserted during translation (e.g., pyrrolysine,
selenocysteine) or made after translation by modifying other amino acids (e.g., diphthamide from histidine). (B)
Disulfide bonds link two cysteine molecules together by their side chains. (C) Glycosylation is the addition of
sugar residues to the surface of proteins. (D) Myristoylation, acetylation, and phosphorylation all add different
groups to amino acids within a protein. These added groups alter the function of a protein. Myristoylation
adds fatty acid chains that tether a protein to the cell membrane. Acetylation and phosphorylation often
activate or deactivate proteins.
Heterologous protein production in yeast
(Saccharomyces cerevisiae)
Advantages
Most are GRAS (generally regarded as safe) listed
Proven history of use in many biotechnological processes
Fermentation technology well established
Ability to carry out posttranslational modifications of
recombinant proteins
Disadvantages
Recombinant proteins usually expressed at low levels
Retention of many exported proteins in the periplasmic space
Adverse public perception of products produced by
recombinant methodologies
Some post-translational modifications differ significantly
from those in animal cells
Heterologous protein production in fungi (Pichia pastoris).
EXPRESSION OF PROTEINS BY YEAST
The yeast genome has been sequenced and many genes have been characterized.
From the viewpoint of biotechnology, several other factors favor the use of yeast for
production of recombinant proteins:
[a) Yeast can be grown easily on both a small scale or in large bioreactors.
[b) After many years of use in brewing and baking, yeast is accepted as a safe organism.
It can therefore be used to produce pharmaceuticals for use in humans without
needing extra government approval.
(c) Yeast normally secretes very few proteins. Consequently, if it is engineered to
release a recombinant protein into the culture medium, this can be purified relatively
easily.
(dl DNA may be transformed into yeast cells either after degrading the cell wall
chemically or enzymatically, or, more usually, by electroporation (see Chapter 3).
[e) A naturally occurring plasmid, the 2-micron circle, is available as a starting point for
developing cloning plasmids.
[f) A variety of yeast promoters have been characterized that are suitable for
driving expression of cloned genes.
[g) Although only a primitive single-celled organism, yeast nonetheless carries out many
of the posttranslational modifications typical of eukaryotic cells, such as addition of
sugar residues (glycosylation). However, yeast only glycosylates proteins that are
secreted.
Vectors for protein
expression are widely available, most being based on a small multi-copy
plasmid (called the 2µ plasmid) which is naturally occurring in this organism.
Such vectors are called YEps (for yeast episomal plasmids), and an
example of one of these is shown in Figure 9.9.
Selection for the presence of the vector is generally done by using an auxotrophic
mutant as the host strain (that is, one which requires a particular nutrient to be
added to the growth medium in order to grow).
In general, eukaryotic expression vectors have the same kinds of features as their
prokaryotic counterparts:
•A
selectable eukaryotic marker gene
• A eukaryotic promoter sequence
• The appropriate eukaryotic transcriptional and
translational stop signals
• A sequence that signals polyadenylation of the
transcript messenger RNA (mRNA)
If the vector is to be used in plasmid form (i.e., as extrachromosomal replicating DNA),
then it must also have an origin of replication that functions in the host cell.
Alternatively, if the vector is designed for integration into the host chromosomal
DNA, then it must carry a sequence that is complementary to a segment of host
chromosomal DNA (the chromosomal integration site) to facilitate recombination.
Yeast artificial chromosome
EXPRESSION OF PROTEINS BY INSECT CELLS
Mammalian cells are relatively delicate and have complex nutritional requirements. This
makes them both difficult and expensive to grow in culture compared to
bacteria or yeasts. However, cultured insect cells are relatively robust and can be grown
in simpler media than mammalian cells. Consequently, insect-based expression systems
have been developed. These have the additional advantage of providing
posttranslational modifications that are very similar to those found in mammalian cells.
The vectors used in cultured insect cells are almost all derived from a family of viruses,
baculoviruses
the
, which infect only insects (and related invertebrates such
as arachnids and crustaceans).
Baculoviruses are unusual in forming packages of virus particles, known as
polyhedrons.
Some infected cells release single virus particles that can infect neighboring cells within
the same insect.
But when the host insect is dead or dying, packages of virus particles embedded in a
protein matrix are released instead.
as polyhedrin
The matrix protein is known
, and the polyhedron structure
protects the virus particles while they are outside the host organism in the environment.
When swallowed by another insect, the polyhedrin is dissolved by digestion and the
polyhedron falls apart. This releases individual virus particles that can infect the cells of
the new host insect.
The polyhedrin gene has an extremely strong promoter, and late during infection, the
polyhedrin protein is made in massive amounts.
Because polyhedrin is not actually needed for virus infection of cultured insect cells, the
polyhedrin promoter may be used to express recombinant proteins. The polyhedrin
coding sequence is removed and replaced by cDNA encoding the protein to be
expressed. There are many different types of baculoviruses, and the one most often
used is multiple nuclear polyhedrosis virus (MNPV). This
infects many insects and replicates well in many cultured insect cell lines. A popular cell
line used to propagate this baculovirus is from the fall armyworm (Spodoptera
frugiperda). Yields of polyhedrin and therefore of a recombinant protein using the
polyhedrin promoter-are especially high in this cell line.
Because the expression vector is a virus, construction is carried out in two stages
(Fig. 10.10). In the first stage, a "transfer" vector is used to carry the cDNA
version of the cloned gene. The transfer vector is an E. coli plasmid that carries a
segment of MNPV DNA, which initially included the polyhedrin gene and flanking
sequences. The polyhedrin coding sequence was then replaced with a multiple cloning
site. When the cloned gene is inserted, it is under control of the polyhedrin promoter.
Construction up to this point is done in E. coli.
In the second stage, the segment containing the cloned gene is recombined onto the
baculovirus, thus replacing the polyhedrin gene. To achieve this, insect cells are
transfected with both the transfer vector and with Vector
MNPV virus DNA. A double-crossover event generates the required recombinant
Bacmids are primarily baculovirus
FIGURE 10.10
Baculovirus Expression Vector To express a gene in insect cells, the gene must be inserted into a
baculovirus genome. First, the gene of interest is cloned into a transfer vector containing the
baculovirus polyhedrin gene promoter followed by a multiple cloning site and the polyhedrin terminator.
This is done in E. coli. The construct is transfected into insect cells along with the normal baculovirus
genome. A double cross-over between the polyhedrin promoter and terminator replaces the polyhedrin
gene with the gene of interest.
One possibility is a shuttle vector that replicates as a plasmid multiple cloning site, and
in E. coli and as a virus in insect cells. Such baculovirus-plasmid hybrids are referred to
as bacmids (Fig. 10.11). They consist of an almost entire baculovirus genome into
which a segment of DNA from an E. coli plasmid has been inserted. This region carries
a bacterial origin of replication, a selective marker, and a multiple cloning site. The
inserted segment replaces the polyhedrin gene of the baculovirus. The cloned gene is
inserted into the MCS giving a recombinant bacmid. Bacmid DNA is then purified from E.
coli and transfected into insect cells, where it replicates DNA of bacterial ori as a virus.
FIGURE 10.11
Bacmid Shuttle Vector Bacmids are primarily baculovirus genomes with the addition of a bacterial
origin of replication, a multiple cloning site, and an antibiotic resistance gene. These sequences allow
the bacmid to survive in E. coli but still infect insect cells.
PROTEIN GLYCOSYLATlON
FIGURE 10.12
Protein Glycosylation Pathways
Mammals and insects share several steps in the pathway for posttranslational glycosylation of
asparagine residues in proteins. However, the final modifications differ as shown. In particular,
mammals add sialic acid residues to the ends of the glycosyl chains, whereas insects do not.
Protein purification from cell culture systems.
Cultured insect cells (Baculovirus vector)
Advantage
Possess many protein processing mechanisms required for higher
eukaryotic proteins
Safe, since very few arthropods are adequate hosts for
baculovirus
Baculovirus vector has received FDA approval for recombinant
protein clinical trial
Few differences in functional and antigenic properties of
recombinant products
Viral infection of cultured cell stops host protein production but
foreign gene is expressed at very high level
Recombinant proteins easy and cost effective to purify
Disadvantage
Information lacking on glycosylation mechanisms
Recombinant protein not always 100% active
Mammalian cells
Advantage
Same biological activity as natural protein
Mammalian expression vectors are commercially available
Mammalian cells can now be grown in large scale culture
Disadvantage
Cells can be difficult to grow
Expensive
Cells grow slowly
Manipulated cells can be genetically unstable
Low productivity of recombinant protein compared to
microorganism
EXPRESSION OF PROTEINS BY MAMMALIAN CELLS
FIGURE 10.13
Mammalian Shuttle VectorMammalian shuttle vectors contain an origin of replication and an antibiotic
resistance gene for growth in bacteria. The vector has a multiple cloning site between a strong
promoter and a polyadenylation signal as well as a eukaryotic origin of replication and a eukaryotic
selection gene, such as npt.
Vectors for introducing DNA into mammalian cells in culture
Components of mammalian plasmid expression vectors
Prokaryotic plasmid sequences
Almost all mammalian expression vectors contain sequences derived from the plasmid
pBR322.
replicon
antibiotic resistance
unique restriction endonuclease sites
The transcriptional unit
The transcriptional unit comprises:
sequences encoding the foreign protein
sequences responsible for driving expression of the gene of
interest (promoter)
intron sequences
mRNA polyadenylation signals.
In the simplest vector, these sequences are provided by a genomic fragment
containing a complete copy of the gene of interest. However, since many mammalian
transcriptional control sequences (promoters and enhancers or chromatin structure)
are to some extent tissue-specific, expression will be possible only in a limited number
of cell types.
More commonly the foreign protein is expressed from a cDNA inserted into a vector
containing the other required components of the transcriptional unit.
Promoter elements
The promoter elements commonly used in mammalian expression vectors include the
SV40 early promoter,
the Rous sarcoma virus (RSV) promoter,
the adenovirus major late promoter, or the
human cytomegalovirus (CMV) immediate early promoter.
Since the inclusion of enhancer sequences can increase the transcriptional activity of
the promoter by 10- to 100-fold, most expression vectors include a strong enhancer
such as those derived from SV40, RSV, or CMV, which are active in a wide variety of
cell types from many species.
In animal cells, the DHFR gene, encoding dihydrofolate reductase, is
sometimes used. This enzyme is required for synthesis of the essential cofactor folic
acid and is inhibited by methotrexate. Mammalian cells lacking the DHFR gene are
used, and a functional copy of the DHFR gene is provided on the shuttle vector.
Treatment with methotrexate inhibits DHFR and hence selects for high-level expression
of the DHFR gene on the vector. Methotrexate levels can be gradually increased, which
selects for a corresponding increase in copy number of the vector. (The chromosomal
DHFR genes must be absent to avoid selecting chromosomal duplications rather than
the vector.)
An alternative approach
An alternative approach is to use a metabolic gene as a dominant selective marker.
The enzyme glutamine synthetase protects cells against the toxic analog, methionine
sulfoximine. The resistance level depends on the copy number of the glutamine
synthetase gene. Therefore, multicopy plasmids carrying the glutamine synthetase gene
can be selected even in cells with functional chromosomal copies of this gene.
Transfection Technologies
Physical Methods
Direct microinjection
Electroporation
biolistic particle delivery.
Biological methods
Virus-mediated transfection
EXPRESSION OF MULTIPLE SUBUNITS
IN MAMMALlAN CELLS
In this case it may be necessary t o synthesize more than one polypeptide in the
same cell (Fig. 10.14). This may be done in three main ways:
[a) Two separate vectors are used, each carrying the gene for one of the subunits.
[b) A single vector is used that carries two separate genes for the two subunits,
each under control o f its own promoter.
(c) A single vector is used that carries an artificial “operon” in which the two genes
are expressed using the same promoter. Transcription gives a single polycistronic
mRNA carrying both genes. However, certain animal viruses have sequences known
as internal ribosomal entry sites (IRESs) that
allow the translation of multiple coding sequencing on the same message.
Expression of Multiple
Polypeptides in the Same
CellSome proteins have two
subunits that assemble
properly only when inside a
mammalian cell. These can be
expressed on one shuttle
vector with two different
promoters (blue), two multiple
cloning sites, and two
polyadenylation sites (purple).
After transfection into a
mammalian cell, both proteins
are expressed. The two protein
subunits assemble using the
necessary cellular components.
Alternatively, the vector may
carry the two genes separated
by an internal ribosome entry
site (IRES), which allows
ribosomes to bind to the
mRNA upstream of both genes.
As before, the two proteins are
made and associate.
FIGURE 10.14
FIGURE 10.15
Comparison of Recombinant Protein Expression SystemsEach protein expression system falls on a
continuum of worst to best for characteristics such as speed, cost, glycosylation, folding, and government
regulations. Transgenic animals (rabbit) and transgenic plants (plants) are discussed in <ce:intra-refstext>Chapters 14 and 15</ce:intra-refs-text><ce:intra-ref-end xlink:href="pii:B978-0-12-175552-2.000142"><ce:intra-ref-title>chapter 14</ce:intra-ref-title></ce:intra-ref-end><ce:intra-ref-end xlink:href="pii:B978-012-175552-2.00015-4"><ce:intra-ref-title>chapter 15</ce:intra-ref-title></ce:intra-ref-end><ce:intra-refs-link />.
The other symbols include mammalian cultured cells, insect cell culture, yeast, and bacteria.