Download Expression of Cloned Gene

2
Production of Protein from
Cloned Genes
Now that we have covered the basic techniques involved in gene cloning and DNA
analysis and examined how these techniques are used in research, we can move on
to consider how recombinant DNA technology is being applied in biotechnology. This
is not a new subject, although biotechnology has received far more attention during
recent years than it ever has in the past. Biotechnology can be defined as the use of
biological processes in industry and technology.
2.1
Overview of Gene Expression
For practical applications it is essential that systems be available in which the cloned
genes can be expressed. Organisms have complex regulatory systems, and many
genes are not expressed all of the time. One of the major goals of genetic
engineering is the development of vectors in which high levels of gene expression
can occur. An expression vector is a vector, which not only can be used to clone the
desired gene but also contains the necessary regulatory sequences so that
expression of the gene is kept under control of the genetic engineer. Some of the
elements involved in gene expression are summarized in the Figure 2.1.
Repressor
binding site
DNA
Anti-leader
Coding sequence
TAC
Promoter
RNA
polymerase
binding site
Operator
ACT
Shine-Delgarno sequence
mRNA
Anti-trailer
Terminator
Transcription
AUG
UGA
Leader (5’-UTR)
Trailer (3’-UTR)
Translation
Protein
H2N
COOH
Figure 2.1: Factors affecting the expression of cloned genes in bacteria. Sequences and
signals that must be appropriate for high levels of gene expression are indicated.
1
2.2
Regulation of Gene Expression
2.2.1
Importance of Regulation of Gene Expression
For maximum production of a protein from a cloned gene, it is usually undesirable to
design a vector that permits the gene to be transcribed and translated at all times.
There are several reasons for this. (i) Some proteins that are of commercial interest
are toxic to the bacterial hosts. (ii) In addition, some expression system, such as
that involving the T7 promoter, are so powerful that normal host genes cannot be
expressed. In either of these cases, it is very desirable that the synthesis of the
protein be under the direct control of the experimenter. The ideal situation is to be
able to grow the culture containing the expression vector until a large population of
cells is obtained, each containing a large copy number of the vector, and then turn
on expression in all copies simultaneously by manipulation of a regulatory switch.
2.2.2
Repressor-Operator System
Recall the major importance of the repressor/operator system in regulating gene
transcription. A strong repressor can completely block the synthesis of the proteins
under its control by binding to the operator region. Repressor function can be turned
off at the chosen time by adding an inducer, allowing the transcription of the genes
controlled by the operator.
For the repressor-operator system to work as a regulatory switch for the production
of a foreign protein, it is desirable to retain in the expression vector a fragment of
the structural gene and the operator controlled by the repressor, to which the cloned
gene is fused. This permits proper arrangement of the sequence of genetic elements:
promoteroperatorribosome-binding
sitestructural
gene,
so
that
efficient
transcription and translation can occur. The presence of a fragment of the normal
protein can help render the foreign protein stable and capable of being excreted.
The construction of plasmid expression vectors containing the regulatory components
of the lac operon provides one means of providing a suitable regulatory switch. The
lac operon is switched on by inducers such as lactose or related -galactosoidase.
Phasing of cell growth and protein synthesis can thus be achieved by allowing growth
to proceed in the absence of inducer until a suitable cell density is achieved, and the
adding inducer (Isopropyl thiogalactoside) to bring about synthesis of the desired
proteins. Plasmids have been constructed containing the lac promoter, ribosome-
2
binding site, and operator. When the desired gene is inserted into such a system,
expression can then be achieved by adding lac inducer.
Another regulatory system that has been used to construct expression vectors is the
tryptophan operon. Although repressed by tryptophan, the trp operon can be induced
by adding a tryptophan analog (e.g., -indolacrylic acid), which brings about an
apparent tryptophan deficiency.
The bacteriophage lambda promoter P L is also sometimes used in expression
systems. This promoter is kept turned off by having the lambda repressor protein in
the cell. Typically the lambda repressor is encoded by a mutant gene (carried by the
vector or by a prophage in the host) and is temperate sensitive. By raising the
temperature of the culture to the proper value (usually 8 to 10C higher than the
growth temperature), the lambda repressor is inactivated and transcription from P L
begins.
Using such expression systems, one can produce very high levels of foreign proteins
in E. coli. In many cases the desired protein exceeds 100,000 molecules per cell and
makes up over 20 percent of the protein molecules in a cell.
2.3
Requirements for Gene Expression
Many factors influence the level of expression of a gene, and a vector must be
constructed in which all these factors are under control. In addition, a host must be
used in which the expression vector is most effective. The key requirements of a
good expression system are summarized here.
2.3.1
Number of Copies of the Gene Per Cell
In general, more product is made if many copies of the gene are present. Vectors
such as small plasmids (e.g., pBR322) are valuable because they can replicate to a
high copy number. Sometimes, often for research purposes, it is desirable to have
only a single copy of the cloned gene in the cell. For these cases, integrating vectors
have been developed so that the gene can recombine into the host chromosome.
2.3.2
Strength of the Transcriptional Promoter
The promoter region is the site at which binding of RNA polymerase first occurs, and
native promoters in different genes vary considerably in RNA polymerase binding
3
strength. For engineering a practical system, it is important to include a strong
promoter in the expression vector. For bacteria, the DNA region around 10 and 35
nucleotides before the start of transcription (called the 10 and 35 region) is
especially important in the promoter. Many Escherichia coli genes are controlled by
relatively weak promoters, and promoters from eukatotes and some other
prokaryotes function poorly or not at all in E. coli. Strong E. coli promoters that have
been used in the construction of expression vectors include lacuv 5 (which normally
controls -galactosidase), try (which normally controls tryptophan synthetase), tac
(a synthetic hybrid of the 35 region of the try promoter and the 10 region of the
lac promoter), lambda PL (which normally regulates lambda virus production), and
ompF (which regulates production of an outer membrane protein).
A novel regulatory system has been created using the bacteriophage T7 promoter
and RNA polymerase. When T7 infects E. coli it codes for its own RNA polymerase,
which
recognizes
only
T7
promoters,
thus
effectively
shutting
down
host
transcription. In expression vectors it is possible to place expression of cloned genes
under control of a T7 promoter. However, if this is done it is necessary to engineer
into the plasmid the gene for T7 RNA polymerase as well. The latter is placed under
control of an easily regulated promoter such as that of lambda or lac. Expression of
the cloned gene(s) occurs shortly after T7 RNA polymerase transcription has been
switched on. Because it recognizes only T7 promoters, T7 RNA polymerase
transcribes only the cloned genes; all other host genes remain untranscribed.
2.3.3
Presence of the Bacterial Ribosome-Binding Site
The transcribed mRNA must bind firmly to the ribosome if translation is to begin, and
an early part of the transcript contains the ribosome-binding site (Shine-Dalgarno
sequence). Bacterial ribosome-binding sites are not found in eukaryotic genes, and it
is thus essential that the bacterial region be present in the cloned gene if high levels
of gene expression are to be obtained. Part of the requirement for proper ribosome
binding is the necessity for a proper distance between the ribosome-binding site and
the translation initiation codon. If these sites are too close or too far apart, the gene
will be translated at low efficiency.
4
2.3.4
Proper Reading Frame
In some cases, the ribosome-binding site and even the initiation codon for the gene
to be cloned are part of the expression vector. Because of the way the source DNA is
fused into such a vector, three possible reading frames could be obtained, only one
of which is satisfactory. One approach that can be used if the correct frame is not
known is the use of three vectors, each having the restriction site into which new
DNA will be inserted positioned such that the insert will be in a different reading
frame. The gene fragment is inserted into all three vectors and the one which gives
proper expression is selected by testing.
2.3.5
Codon Uses
There is more than one codon for most of the 20 amino acids, and some codons are
used more frequently than others. Codon usage is partly a function of the
concentration of the appropriate tRNA in the cell. A condon frequently used in a
mammalian cell may be used less frequently in the organism in which the gene is
being cloned. Insertion of the appropriate codon would be difficult because it would
have to be changed in all locations in the gene. However, this can be done if
necessary by using synthetic DNA and site-directed mutagenesis to create a gene
more amenable to the codon usage patterns of the host.
2.3.6
Fate of the Protein after It is Produced
Some proteins are susceptible to degradation by intracellular proteases and may be
destroyed before they can be isolated. Excreted proteins must have the signal
sequence attached if they are to move through the cytoplasmic membrane. Some
eukaryotic proteins are toxic to the prokaryotic host, and the host for the cloning
vector may be killed before a sufficient amount of the product is synthesized. Further
engineering of either the host or the vector may be necessary to eliminate these
problems. The skill of the genetic engineer is thus essential in the construction of an
appropriate vector, which can be (1) efficiently incorporated into the proper host, (2)
replicated to high copy number, (3) efficiently transcribed, and (4) efficiently
translated. Many mammalian proteins are completely unexpressed when their genes
are first cloned in E. coli, but expression can sometimes be achieved with appropriate
manipulation of the vector. The best example is the production on a commercial
scale of human insulin in E. coli.
5
2.4
Expression Vector
A cloning vector that has been constructed in such a way that, after insertion of a
DNA molecule, its coding sequence is properly transcribed and the mRNA is
translated. The cloned gene is put under the control of a promoter sequence for the
initiation of transcription, and often also has a transcription termination sequence
at its end.
2.4.1
Choice of Expression System
After you have decided which protein or which domain(s) of a protein you would like
to clone and express, you have to think about which expression system you would
like to use. At present there are many different expression systems available (Table
2.1):
Table 2.1: Comparison of expression systems
Characteristics
E. coli
Yeast
Insect cells
Mammalian cells
Cell growth
Rapid
(30 min)
Rapid
(90 min)
Slow
(18-24 h)
Slow
(24 h)
Complexity of growth
medium
Minimum
Minimum
Complex
Complex
Cost of growth medium
Low
Low
High
High
Expression level
High
Low - High
Low - High
Low - Moderate
Secretion to
periplasm
Secretion to
medium
Secretion to
medium
Secretion to
medium
Refolding
usually required
Refolding
may be
required
Proper
folding
Proper
folding
N-Linked glycosylation
None
High
mannose
Simple,
no sialic acid
Complex
O-Linked glycosylation
No
Yes
Yes
Yes
Phosphorylation
No
Yes
Yes
Yes
Acetylation
No
Yes
Yes
Yes
Acylation
No
Yes
Yes
Yes
-Carboxylation
No
No
No
Yes
Extracellular expression
Posttranslational modifications
Protein folding
(1)
Prokaryotic expression systems: (i) Escherichia coli, (ii) Lactococcus lactis, and
(iii) Other bacteria, e.g. Bacillus species.
(2)
Yeast: (i) Pichia pastoris, (ii) Pichia methanolica, and (iii) Saccharomyces
cerevisiae.
6
(3)
Insect cells: (i) Baculovirus, (ii) Stable recombinant cell lines (e.g. Insect select
system - Invitrogen), and (iii) Schneider cells (Drosophila).
(4)
Mammalian cells: (i) Viral infection, e.g., Adenovirus, Semliki forest virus, (ii)
Inducible expression systems.
(5)
In vitro expression systems: (ii) Rabbit reticulocyte lysate (red blood cells), (ii)
Wheat germ extract, and (iii) Escherichia coli extract.
(6)
Others: (i) Xenopus oocytes and cell-free extract, (ii) Transgenic mice, (iii) Milk
of transgenic animals, and (iv) Transgenic plants.
2.4.2
Choice of Expression System
To determine which system is the best choice, ask yourself the following questions:
1. What type of protein do I want to express?
When you would like to express a protein of prokaryotic origin, the obvious choice is
to use E. coli. The method is quick and cheap and the organism has all the
machinery necessary for folding and post-translational modifications. In case the
protein is from a eukaryotic source, the method of choice will depend on more
factors (see below).
2. Do I get soluble protein when I express in E. coli?
Also for the expression of eukaryotic proteins the first method of choice is normally
E. coli for the above mentioned reasons. However, many eukaryotic proteins don't
fold properly in E. coli and form insoluble aggregates (inclusion bodies). Sometimes
it is possible to resolubilize the protein from the inclusion bodies or improve the
solubility by expressing the protein at a lower temperature. Also expression of your
target protein as a fusion protein with a highly soluble partner such as glutathione-Stransferase (GST), maltose binding protein (MBP), or DsbA can improve its solubility.
Often, however, it is better to change to an eukaryotic expression system because it
is better equipped to fold proteins from an eukaryotic source. Thus, instead of trying
out 10 different E. coli constructs, it is better to switch expression system.
3. Does my protein need post-translational modifications for structure or
activity?
Many proteins need to be modified following translation in order to become active
and/or adapt the proper structure. The simplest of these modifications is the removal
of the N-terminal methionine residue, which can occur in all organisms. More
7
complex modifications, like N- and O-glycosylation, phosphorylation, are exclusively
carried out by eukaryotic cells. Keep in mind that not all eukaryotic cells carry out
the same modifications. Check Table 2.1 to find out which expression system carries
out the post-translational modification(s) you are looking for.
4. What is the codon usage in my protein?
Not all of the 61 mRNA codons are used equally. The so-called major codons are
those that occur in highly expressed genes, whereas the minor or rare codons tend
to be in genes expressed at a low level. Which of the 61 codons are the rare ones
depends strongly on the organism. The codon usage per organism can be found in
the Codon Usage Database. Usually, the frequency of the codon usage reflects the
abundance of their cognate tRNAs. Therefore, when the codon usage of the protein
you would like to express differs significantly from the average codon usage of the
expression host, this could cause problems during expression. The following
problems are often encountered:
(i)
Interrupted translation, which leads to a variety of truncated protein
products,
(ii)
Frame shifting,
(iii) Misincorporation of amino acids. For instance, lysine for arginine as a result
of the AGA codon, and
(iv) Inhibition of protein synthesis and cell growth
As a consequence, the observed levels of expression are often low or there will be no
expression at all. Especially in cases where rare codons are present at the 5'-end of
the mRNA or where consecutive rare codons are found expression levels are low and
truncated protein products are found.
To increase the expression levels of proteins containing rare codons in E. coli, two
main methods are available:
(i)
Site-directed mutagenesis to replace the rare codons by more commonly
used codons for the same residue; e.g. the rare argenines codons AGA and
AGG by the E. coli preferred CGC codon.
(ii)
Co-expression of the genes, which encode rare tRNAs. There are several
commercial E. coli strains available that encode for a number of the rare codon
genes.
8
Often you will obtain a mixture of full-length protein and truncated species. Providing
the protein with a C-terminal tag (e.g. His6-tag) will help you to purify only the fulllength protein using affinity chromatography. When both above-mentioned methods
fail to increase expression levels, it is time to change expression system and try to
express your protein in yeast or insect cells. In cases where the protein contains
many rare E. coli codons it is probably better to immediately start with a eukaryotic
system.
2.5
Escherichia coli Expression Vector Features
If a foreign (i.e., non-bacterial) gene is simply ligated into a standard vector and
cloned in E. coli, it is very unlikely that a significant amount of recombinant protein
will be synthesized. This is because expression is dependent on the gene being
surrounded by a collection of signals that can be recognized by the bacterium. These
signals, which are short sequences of nucleotides, advertise the presence of the gene
and provide instructions for the transcriptional and translational apparatus of the
cell. The basic architecture of an E. coli expression vector is shown in Figure 2.2
and contains the following features:
Figure 2.2: Basic architecture of an E. coli expression vector.
9
2.5.1
Selectable Marker
In the absence of selective pressure plasmids are lost from the host. Especially in the
case of very high copy number plasmids and when plasmid-borne genes are toxic to
the host or otherwise significantly reduce its growth rate. The simplest way to
address this problem is to express from the same plasmid an antibiotic-resistance
marker and supplement the medium with the appropriate antibiotic to kill plasmidfree cells. The most used antibiotics and their effective concentrations are listed in
Table 2.2.
Table 2.2: Most used antibiotics and their effective concentrations
Concentration (g/ml)
Antibiotic
Amp
Ampicillin
100
Cab
Carbenicillin
100
Cam
Chloramphenicol
34
Kan
Kanamycin
30
Rif
Rifampicin
200
Spc
Spectinomycin
Tet
Tetracyclin
2.5.2
50
12.5
Regulatory Gene (Repressor)
Many promoters show leakiness in their expression i.e. gene products are expressed
at low level before the addition of the inducer. This becomes a problem when the
gene product is toxic for the host. This can be prevented by the constitutive
expression of a repressor protein.
The lac-derived promoters are especially leaky. These promoters can be controlled
by the insertion of a lac-operator sequence downstream the promoter and the
expression of the lac-repressor by host strains carrying the lacIq allele (for medium
copy number plasmids) or from the same or a helper plasmid (for higher copy
number plasmids). Alternatively, repression can be achieved by the addition of 1%
glucose to the culture medium.
2.5.3
Origin of Replication
The origin of replication controls the plasmid copy number.
10
2.5.4
Promoter
The promoter is the most important component of an expression vector. This is
because the promoter controls the very first stage of gene expression (attachment of
an RNA polymerase enzyme to the DNA) and determines the rate at which mRNA is
synthesized. The amount of recombinant protein obtained therefore depends to a
great extent on the nature of the promoter carried by the expression vector. The
promoter initiates transcription and is positioned 10-100 nucleotides upstream of the
ribosome-binding site. The ideal promoter exhibits several desirable features:
(i)
It is strong enough to allow product accumulation up to 50% of the total
cellular protein.
(ii)
It has a low basal expression level (i.e., it is tightly regulated to prevent
product toxicity).
(iii) It is easy to induce.
2.5.4.1 Examples of promoters used in expression vectors
Several E. coli promoters combine the desired features of strength and ease of
regulation. Those most frequently used in expression vectors are as follows:
(i)
The lac promoter (Figure 2.3a) is the sequence that controls transcription of
the lacZ gene coding for -galactosidase (and also the lacZ’ gene fragment
carried by the pUC and M13mp vectors. The lac promoter is induced by
isopropylthiogalactoside (IPTG), so addition of this chemical into the growth
medium switches on transcription of a gene inserted downstream of the lac
promoter carried by an expression vector.
(ii)
The trp promoter (Figure 2.3b) is normally upstream of the cluster of genes
coding for several of the enzymes involved in biosynthesis of the amino acid
tryptophan. The trp promoter is repressed by tryptophan, but is more easily
induced by 3--indoleacrylic acid.
(iii) The tac promoter (Figure 2.3c) is a hybrid between the trp and lac
promoters. It is stronger than either, but still induced by IPTG.
(iv) The PL promoter (Figure 2.3d) is one of the promoters responsible for
transcription of the  DNA molecule. PL is a very strong promoter that is
recognized by the E. coli RNA polymerase, which is subverted by  into
transcribing the bacteriophage DNA. The promoter is repressed by the product
of the cI gene. Expression vectors that carry the PL promoter are used with a
mutant E. coli host that synthesizes a temperature-sensitive form of the cI
11
protein. At a low temperature (less than 30°C) this mutant cI protein is able to
repress the PL promoter, but at higher temperatures the protein is inactivated,
resulting in transcription of the cloned gene.
(v)
The T7 promoter (Figure 2.3e) is specific for the RNA polymerase coded by
T7 bacteriophage. This RNA polymerase is much more active than the E. coli
RNA polymerase, which means that a gene inserted downstream of the T7
promoter will be expressed at a high level. The gene for the T7 RNA polymerase
is not normally present in the E. coli genome, so a special strain of E. coli is
needed, one which is lysogenic for T7 phage. Remember that a lysogen
contains an inserted copy of the phage DNA in its genome. In this particular
strain of E. coli, the phage DNA has been altered by placing a copy of the lac
promoter upstream of its gene for the T7 RNA polymerase. Addition of IPTG to
the growth medium therefore switches on synthesis of the T7 RNA polymerase,
which in turn leads to activation of the gene carried by the T7 expression
vector.
Figure 2.3: Five promoters frequently used in expression vectors. The lac and trp promoters
are shown upstream of the genes that they normally control in E. coli.
12
2.5.5
Transcription Terminator
The terminator, which marks the point at the end of the gene where transcription
should stop. A terminator is usually a nucleotide sequence that can base pair with
itself to form a stem–loop structure. The transcription terminator reduces unwanted
transcription and increases plasmid and mRNA stability.
2.5.6
Shine-Delgarno Sequence
The Shine-Dalgarno (SD) sequence is required for translation initiation and is
complementary to the 3'-end of the 16S ribosomal RNA. The efficiency of translation
initiation at the start codon depends on the actual sequence. The concensus
sequence is: 5'-TAAGGAGG-3'. It is positioned 4-14 nucleotides upstream the start
codon with the optimal spacing being 8 nucleotides. To avoid formation of secondary
structures (which reduces expression levels) this region should be rich in A residues.
2.5.7
Start Codon
Initiation point of translation. In E. coli the most used start codon is ATG. GTG is
used in 8% of the cases. TTG and TAA are hardly used.
2.5.8
Tags and Fusion Proteins
N- or C-terminal fusions of heterologous proteins to short peptides (tags) or to other
proteins (fusion partners) offer several potential advantages (Table 2.3):
(i)
Improved expression. Fusion of the N-terminus of a heterologous protein to
the C-terminus of a highly-expressed fusion partner often results in high level
expression of the fusion protein.
(ii)
Improved solubility. Fusion of the N-terminus of a heterologous protein to
the C-terminus of a soluble fusion partner often improves the solubility of the
fusion protein.
(iii) Improved detection. Fusion of a protein to either terminus of a short peptide
(epitope tag) or protein, which is recognized by an antibody, or a binding
protein (Western blot analysis) or by biophysical methods (e.g. GFP by
fluorescence)
allows
for
detection
of
a
protein
during
expression
and
purification.
(iv) Improved purification. Simple purification schemes have been developed for
proteins fused at either end to tags or proteins, which bind specifically to
affinity resins.
13
Table 2.3: Tags and fusion partners
Tag or fusion partner
Size
Location
His-tag
6 or
10 aa
N, C,
internal
Expression Secretion Purification Detection
X
X
Avidin
X
X
X
Streptavidin
X
X
X
X
X
Streptococcal protein G
28 kDa
N, C
X
Glutathione-S-transferase
(GST)
26 kDa
N
X
Maltose-binding domain (MBP)
40 kDa
N, C
X
Green fluorescent protein
(GFP)
220 aa
N, C
Disulfide oxidoreductase
(DsbA)
208 aa
N
2.5.9
X
X
X
X
X
X
Protease Cleavage Site
Protease cleavage sites are often added to be able to remove a tag or fusion partner
from the fusion protein after expression. Most commonly used proteases are listed in
Table 2.4. However, cleavage is rarely complete and often additional purification
steps are required.
Table 2.4: Protease cleavage sites
Protease
Recognition sequence
Source
Factor Xa
Ile Glu/Asp Gly Arg |
Different distributors
Enterokinase
Asp Asp Asp Asp Lys |
New England Biolabs
Thrombin
Leu Val Pro Arg | Gly Ser
Different distributors
TEV protease
Glu Asn Leu Tyr Phe Gln | Gly
Invitrogen
2.5.10 Multiple Cloning Sites
A series of unique restriction sites that enables you to clone your gene of interest
into the vector.
2.5.11 Stop Codon
Termination of translation. There are 3 possible stop codons but TAA is preferred
because it is less prone to read-through than TAG and TGA. The efficiency of
termination is increased by using 2 or 3 stop codons in series.
14
2.6
General Problems with the Production of
Recombinant Protein in E. coli
Despite the development of sophisticated expression vectors, there are still
numerous difficulties associated with the production of protein from foreign genes
cloned in E. coli. These problems can be grouped into two categories: those that are
due to the sequence of the foreign gene, and those that are due to the limitations of
E. coli as a host for recombinant protein synthesis.
2.6.1
Problems Resulting from the Sequence of the Foreign
Gene
There are three ways in which the nucleotide sequence might prevent efficient
expression of a foreign gene cloned in E. coli:
(i)
The foreign gene might contain introns. This would be a major problem, as E.
coli genes do not contain introns and therefore the bacterium does not possess
the necessary machinery for removing introns from transcripts (Figure 2.4a).
(ii)
The foreign gene might contain sequences that act as termination signals in
E. coli (Figure 2.4b). These sequences are perfectly innocuous in the normal
host cell, but in the bacterium result in premature termination and a loss of
gene expression.
(iiii) The codon bias of the gene may not be ideal for translation in E. coli. Although
virtually all organisms use the same genetic code, each organism has a bias
toward preferred codons. This bias reflects the efficiency with which the tRNA
molecules in the organism are able to recognize the different codons. If a
cloned gene contains a high proportion of disfavored codons, the E. coli tRNAs
may encounter difficulties in translating the gene, reducing the amount of
protein that is synthesized (Figure 2.4c).
These problems can usually be solved, although the necessary manipulations may be
time-consuming and costly (an important consideration in an industrial project). If
the gene contains introns then its complementary DNA (cDNA), prepared from the
mRNA and so lacking introns, might be used as an alternative. In vitro mutagenesis
could then be employed to change the sequences of possible terminators and to
replace disfavored codons with those preferred by E. coli. An alternative with genes
that are less than 1 kb in length is to make an artificial version. This involves
synthesizing a set of overlapping oligonucleotides that are ligated together, the
15
sequences of the oligonucleotides being designed to ensure that the resulting gene
contains preferred E. coli codons and that terminators are absent.
Figure 2.4: Three of the problems that could be encountered when foreign genes are
expressed in E. coli: (a) introns are not removed in E. coli; (b) premature termination of
transcription; (c) a problem with codon bias.
16
2.6.2
Problems Caused by E. coli
Some of the difficulties encountered when using E. coli as the host for recombinant
protein synthesis stem from inherent properties of the bacterium. For example:
(i)
E. coli might not process the recombinant protein correctly. The proteins of
most organisms are processed after translation, by chemical modification of
amino acids within the polypeptide. Often these processing events are essential
for the correct biological activity of the protein. Unfortunately, the proteins of
bacteria and higher organisms are not processed identically. In particular, some
animal proteins are glycosylated, meaning that they have sugar groups
attached to them after translation. Glycosylation is extremely uncommon in
bacteria and recombinant proteins synthesized in E. coli are never glycosylated
correctly.
(ii)
E. coli might not fold the recombinant protein correctly, and generally is unable
to synthesize the disulphide bonds present in many animal proteins. If the
protein does not take up its correctly folded tertiary structure, then usually it is
insoluble and forms an inclusion body within the bacterium (Figure 2.5).
Recovery of the protein from the inclusion body is not a problem, but
converting the protein into its correctly folded form can be difficult or
impossible in the test tube. Under these circumstances the protein is, of course,
inactive.
(iii) E. coli might degrade the recombinant protein. Exactly how E. coli can
recognize the foreign protein, and thereby subject it to preferential turnover, is
not known.
Figure 2.5: Inclusion bodies.
17
These problems are less easy to solve than the sequence problems described in the
previous section. Degradation of recombinant proteins can be reduced by using as
the host a mutant E. coli strain that is deficient in one or more of the proteases
responsible for protein degradation. Correct folding of recombinant proteins can also
be promoted by choosing a special host strain, in this case one that over-synthesizes
the chaperone proteins thought to be responsible for protein folding in the cell. But
the main problem is the absence of glycosylation. So far this has proved
insurmountable, limiting E. coli to the synthesis of animal proteins that do not need
to be processed in this way.
2.7
In Vitro Translation
2.7.1
Application of In Vitro Translation
The in vitro synthesis of proteins in cell-free extracts is an important tool for
molecular biologists and has a variety of applications, including the (1) rapid
identification of gene products (e.g., proteomics), (2) localization of mutations
through synthesis of truncated gene products, (3) protein folding studies, and (4)
incorporation of modified or unnatural amino acids for functional studies.
2.7.2
Advantages of in vitro Translation
The use of in vitro translation systems can have advantages over in vivo gene
expression (1) when the over-expressed product is toxic to the host cell, (2) when
the product is insoluble or forms inclusion bodies, or (3) when the protein undergoes
rapid proteolytic degradation by intracellular proteases.
In principle, it should be possible to prepare a cell-free extract for in vitro translation
of mRNAs from any type of cells. In practice, only a few cell-free systems have been
developed for in vitro protein synthesis. In general, these systems are derived from
cells engaged in a high rate of protein synthesis. This article will explain different
approaches to in vitro protein synthesis (translation of purified RNA versus “linked”
and “coupled” transcription:translation) and will also describe basic differences
between eukaryotic and prokaryotic cell-free systems.
2.7.3
Cell-Free Expression Systems
The most frequently used cell-free translation systems consist of extracts from:
(1) Rabbit reticulocytes, (2) Wheat germ and (3) Escherichia coli.
18
All are prepared as crude extracts containing all the macromolecular components
(70S or 80S ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation, elongation
and termination factors, etc.) required for translation of exogenous RNA. To ensure
efficient translation, each extract must be supplemented with amino acids, energy
sources (ATP, GTP), energy regenerating systems (creatine phosphate and creatine
phosphokinase for eukaryotic systems, and phosphoenol pyruvate and pyruvate
kinase for the E. coli lysate), and other co-factors (Mg2+, K+, etc.).
There are two approaches to in vitro protein synthesis based on the starting genetic
material: RNA or DNA. Standard translation systems, such as reticulocyte lysates
and wheat germ extracts, use RNA as a template; whereas “coupled” and “linked”
systems start with DNA templates, which are transcribed into RNA then translated.
Each of these systems is discussed below.
2.7.3.1 Rabbit Reticulocyte Lysate
Rabbit reticulocyte lysate is a highly efficient in vitro eukaryotic protein synthesis
system used for translation of exogenous RNAs (either natural or generated in vitro).
In vivo, reticulocytes are highly specialized cells primarily responsible for the
synthesis of haemoglobin, which represents more than 90% of the protein made in
the reticulocyte. These immature red cells have already lost their nuclei, but contain
adequate mRNA, as well as complete translation machinery, for extensive globin
synthesis. The endogenous globin mRNA can be eliminated by incubation with Ca 2+dependent micrococcal nuclease, which is later inactivated by chelation of the Ca 2+
by ethyleneglycol tetraacetic acid (EGTA). Ambion offers a nuclease-treated
reticulocyte lysate. This type of lysate is the most widely used RNA-dependent cellfree system because of its low background and its efficient utilization of exogenous
RNAs even at low concentrations (Figure 2.6). Exogenous proteins are synthesized
at a rate close to that observed in intact reticulocyte cells.
Untreated reticulocyte lysate translates endogenous globin mRNA, exogenous RNAs,
or both. This type of lysate is typically used for studying the translation machinery,
e.g. studying the effects of inhibitors on globin translation. Both the untreated and
treated rabbit reticulocyte lysates have low nuclease activity and are capable of
synthesizing a large amount of full-length product. Both lysates are appropriate for
19
the synthesis of larger proteins from either capped or uncapped RNAs (eukaryotic or
viral).
Figure 2.6: Standard in vitro translation procedure using rabbit reticulocyte lysate or wheat
germ extract.
2.7.3.2 Wheat Germ Extract
Wheat germ extract is a convenient alternative to the rabbit reticulocyte lysate cellfree system. This extract has low background incorporation due to its low level of
endogenous mRNA. Wheat germ lysate efficiently translates exogenous RNA from a
variety of different organisms, from viruses and yeast to higher plants and
mammals. The wheat germ extract is recommended for translation of RNA containing
small fragments of double-stranded RNA or oxidized thiols, which are inhibitory to
the rabbit reticulocyte lysate. Both retic and wheat germ extracts translate RNA
isolated from cells and tissue or those generated by in vitro transcription (see Figure
2.6). When using RNA synthesized in vitro, the presence of a 5' cap structure may
enhance translational activity. Typically, translation by wheat germ extracts is more
cap-dependent than translation by retic extracts. If capping of the RNA is impossible
and the protein yield from an uncapped mRNA is low, the coding sequence can be
subcloned into a prokaryotic vector and expressed directly from a DNA template in
an E. coli cell-free system.
20
2.7.3.3 E. coli Cell-Free System
E. coli cell-free systems consist of a crude extract that is rich in endogenous mRNA.
The extract is incubated during preparation so that this endogenous mRNA is
translated and subsequently degraded. Because the levels of endogenous mRNA in
the prepared lysate is low, the exogenous product is easily identified. In comparison
to eukaryotic systems, the E.coli extract has a relatively simple translational
apparatus with less complicated control at the initiation level, allowing this system to
be very efficient in protein synthesis. Bacterial extracts are often unsuitable for
translation of RNA, because exogenous RNA is rapidly degraded by endogenous
nucleases. There are some viral mRNAs (TMV, STNV, and MS2) that translate
efficiently, because they are somewhat resistant to nuclease activity and contain
stable secondary structure. However, E. coli extracts are ideal for coupled
transcription : translation from DNA templates.
2.7.4
Linked and Coupled Transcription:Translation Systems
In standard translation reactions, purified RNA is used as a template for translation.
“linked” and “coupled” systems, on the other hand, use DNA as a template. RNA is
transcribed from the DNA and subsequently translated without any purification. Such
systems typically combine a prokaryotic phage RNA polymerase and promoter (T7,
T3, or SP6) with eukaryotic or prokaryotic extracts to synthesize proteins from
exogenous DNA templates. DNA templates for transcription:translation reactions may
be cloned into plasmid vectors or generated by PCR.
2.7.4.1 Linked Transcription:Translation Systems
The "linked" system is a two-step reaction, based on transcription with a
bacteriophage polymerase followed by translation in the rabbit reticulocyte lysate or
wheat germ lysate (Figure 2.7). Because the transcription and translation reactions
are separate, each can be optimized to ensure that both are functioning at their full
potential.
Conversely,
many
commercially
available
eukaryotic
coupled
transcription:translation systems have compromised one or both reactions so that
they can occur in a single tube. Thus, yield is sacrificed for convenience.
21
Figure 2.7: Linked in vitro transcription and translation procedure using rabbit reticulocyte
lysate.
2.7.4.2 Coupled Transcription:Translation Systems
Unlike eukaryotic systems where transcription and translation occur sequentially, in
E. coli, transcription and translation occur simultaneously within the cell. In vitro E.
coli translation systems are thus performed the same way, coupled, in the same tube
under the same reaction conditions (one-step reaction; Figure 2.8). During
transcription, the 5' end of the RNA becomes available for ribosomal binding and
undergoes translation while its 3' end is still being transcribed. This early binding of
ribosomes
to
the
RNA
maintains
transcript
stability
and
promotes
efficient
translation. This bacterial translation system gives efficient expression of either
prokaryotic or eukaryotic gene products in a short amount of time. For the highest
protein yield and the best initiation fidelity, make sure the DNA template has a
22
Shine-Dalgarno ribosome binding site upstream of the initiator codon. Capping of
eukaryotic RNA is not required. Use of E.coli extract also eliminates cross-reactivity
or other problems associated with endogenous proteins in eukaryotic lysates. Also,
the E. coli S30 extract system allows expression from DNA vectors containing natural
E. coli promoter sequences (such as lac or tac).
Figure 2.8: Coupled in vitro transcription:translation procedure using E. coli extract.
2.8
Important Elements for Translation
There are some significant differences between prokaryotic and eukaryotic mRNA
transcripts.
2.8.1
Typically,
Eukaryotic mRNA Transcripts
eukaryotic
mRNAs
are
characterized
by
two
post-transcriptional
modifications: (i) a 5'-7-methyl-GTP cap and (ii) a 3'-poly(A) tail. Both modifications
contribute to the stability of the mRNA by preventing degradation. Additionally, the
5' cap structure enhances the translation of mRNA by helping to bind the eukaryotic
ribosome and assuring recognition of the proper AUG initiator codon. This function
may vary with the translation system and with the specific mRNA being synthesized.
The consensus sequence 5'-GCCACCAUGG-3', also known as the "Kozak" sequence,
is considered to be the strongest ribosomal binding signal in eukaryotic mRNA. For
23
efficient translation initiation, the key elements are the G residue at the +1 position
and the A residue at the -3 position. An mRNA that lacks the Kozak consensus
sequence may be translated efficiently in eukaryotic cell-free systems if it possesses
a moderately long 5'-untranslated region (UTR) that lacks stable secondary
structure.
2.8.2
Prokaryotic mRNA Transcripts
In bacteria, the ribosome is guided to the AUG initiation site by a purine-rich region
called the Shine-Dalgarno (SD) sequence. This sequence is complementary to the 3'
end of the 16s rRNA in the 30S ribosomal subunit. Upstream from the initiation AUG
codon, the SD region has the consensus sequence 5'-UAAGGAGGUGA-3'. Specific
mRNAs vary considerably in the number of nucleotides that complement the antiShine-Dalgarno sequence of 16S rRNA, ranging from as few as two to nine or more.
The position of the ribosome-binding site (RBS) in relation to the AUG initiator is
very important for efficiency of translation (usually from -6 to -10 relative to the A of
the initiation site).
2.9
Production of Recombinant Protein by
Eukaryotic Cells
The problems associated with obtaining high yields of active recombinant proteins
from genes cloned in E. coli have led to the development of expression systems for
other organisms. There have been a few attempts to use other bacteria as the hosts
for recombinant protein synthesis, and some progress has been made with Bacillus
subtilis, but the main alternatives to E. coli are microbial eukaryotes. The argument
is that a microbial eukaryote, such as a yeast or filamentous fungus, is more closely
related to an animal, and so may be able to deal with recombinant protein synthesis
more efficiently than E. coli. Yeasts and fungi can be grown just as easily as bacteria
in continuous culture, and might express a cloned gene from a higher organism, and
process the resulting protein in a manner more akin to that occurring in the higher
organism itself.
2.9.1
Recombinant Protein from Yeast and Filamentous Fungi
To a large extent the potential of microbial eukaryotes has been realized and these
organisms are now being used for the routine production of several animal proteins.
Expression vectors are still required because it turns out that the promoters and
24
other expression signals for animal genes do not, in general, work efficiently in these
lower eukaryotes.
2.9.1.1 Saccharomyces cerevisiae as the Host for Recombinant
Protein Synthesis
The yeast Saccharomyces cerevisiae is currently the most popular microbial
eukaryote for recombinant protein production. Cloned genes are often placed under
the control of the GAL promoter (Figure 2.9a), which is normally upstream of the
gene coding for galactose epimerase, an enzyme involved in the metabolism of
galactose. The GAL promoter is induced by galactose, providing a straightforward
system for regulating expression of a cloned foreign gene. Other useful promoters
are PHO5, which is regulated by the phosphate level in the growth medium, and
CUP1, which is induced by copper. Most yeast expression vectors also carry a
termination sequence from an S. cerevisiae gene, because animal termination signals
do not work effectively in yeast.
Figure 2.9: Four promoters frequently used in expression vectors for microbial eukaryotes.
P = promoter.
Yields of recombinant protein are relatively high, but (1) S. cerevisiae is unable to
glycosylate
animal
proteins
correctly,
often
adding
too
many
sugar
units
(“hyperglycosylation”), although this can be prevented or at least reduced by using a
mutant host strain. (2) S. cerevisiae also lacks an efficient system for secreting
25
proteins into the growth medium. In the absence of secretion, recombinant proteins
are retained in the cell and consequently are less easy to purify. (3) Codon bias can
also be a problem.
Despite these drawbacks, S. cerevisiae remains the most frequently used microbial
eukaryote for recombinant protein synthesis, partly because it is accepted as a safe
organism for production of proteins for use in medicines or in foods, and partly
because of the wealth of knowledge built up over the years regarding the
biochemistry and genetics of S. cerevisiae, which means that it is relatively easy to
devise strategies for minimizing the difficulties that arise.
7.9.1.2 Other Yeasts and Fungi
Although Saccharomyces cerevisiae retains the loyalty of many molecular biologists,
there are other microbial eukaryotes that might be equally if not more effective in
recombinant protein synthesis. In particular, Pichia pastoris, a second species of
yeast, is able to synthesize large amounts of recombinant protein (up to 30% of the
total cell protein) and its glycosylation abilities are very similar to those of animal
cells. The sugar structures that it synthesizes are not precisely the same as the
animal versions (Figure 2.10), but the differences are relatively trivial and would
probably not have a significant effect on the activity of a recombinant protein.
Importantly, the glycosylated proteins made by P. pastoris are unlikely to induce an
antigenic reaction if injected into the bloodstream, a problem frequently encountered
with the over-glycosylated proteins synthesized by S. cerevisiae.
Expression vectors for P. pastoris make use of the alcohol oxidase (AOX) promoter
(Figure 2.9b), which is induced by methanol. The only significant problem with P.
pastoris is that it sometimes degrades recombinant proteins before they can be
purified, but this can be controlled by using special growth media. Other yeasts that
have been used for recombinant protein synthesis include Hansenula polymorpha,
Yarrowia lipolytica, and Kluveromyces lactis. The last of these has the attraction that
it can be grown on waste products from the food industry.
26
Figure 2.10: Comparison between a typical glycosylation structure found on an animal
protein and the structures synthesized by Pichia pastoris and Saccharomyces cerevisiae.
The two most popular filamentous fungi are Aspergillus nidulans and Trichoderma
reesei. The advantages of these organisms are their good glycosylation properties
and their ability to secrete proteins into the growth medium. The latter is a
particularly strong feature of the wood rot fungus T. reesei, which in its natural
habitat secretes cellulolytic enzymes that degrade the wood it lives on. The secretion
characteristics mean that these fungi are able to produce recombinant proteins in a
form that aids purification. Expression vectors for A. nidulans usually carry the
glucoamylase promoter (Figure 2.9c), induced by starch and repressed by xylose;
those for T. reesei make use of the cellobiohydrolase promoter (Figure 2.9d), which
is induced by cellulose.
2.9.2
Using Animal Cells for Recombinant Protein Production
The difficulties inherent in synthesis of a fully active animal protein in a microbial
host have prompted biotechnologists to explore the possibility of using animal cells
for recombinant protein synthesis. For proteins with complex and essential
glycosylation structures, an animal cell might be the only type of host within which
the active protein can be synthesized.
27
2.9.2.1 Protein Production in Mammalian Cells
Culture systems for animal cells have been around since the early 1960s, but only
during the past 20 years have methods for large-scale continuous culture become
available. A problem with some animal cell lines is that they require a solid surface
on which to grow, adding complications to the design of the culture vessels. (1) One
solution is to fill the inside of the vessel with plates, providing a large surface area,
but this has the disadvantage that complete and continuous mixing of the medium
within the vessel becomes very difficult. (2) A second possibility is to use a standard
vessel but to provide the cells with small inert particles (e.g., cellulose beads) on
which to grow.
Rates of growth and maximum cell densities are much less for animal cells compared
with microorganisms, limiting the yield of recombinant protein, but this can be
tolerated if it is the only way of obtaining the active protein.
Of course, gene cloning may not be necessary in order to obtain an animal protein
from an animal cell culture. Nevertheless, expression vectors and cloned genes are
still used to maximize yields, by placing the gene under control of a promoter that is
stronger than the one to which it is normally attached. This promoter is often
obtained from viruses such as SV40, cytomegalovirus (CMV), or Rous sarcoma virus
(RSV).
Mammalian cell lines derived from humans or hamsters have been used in synthesis
of several recombinant proteins, and in most cases these proteins have been
processed correctly and are indistinguishable from the non-recombinant versions.
However, this is the most expensive approach to recombinant protein production,
especially as the possible co-purification of viruses with the protein means that
rigorous quality control procedures must be employed to ensure that the product is
safe.
2.9.2.2 Protein Production in Insect Cells
Insect cells provide an alternative to mammalian cells for animal protein production.
Insect cells do not behave in culture any differently to mammalian cells but they
have the great advantage that, thanks to a natural expression system, they can
provide high yields of recombinant protein.
28
The Expression System Based on Baculoviruses
The expression system is based on the baculoviruses, a group of viruses that are
common in insects but do not normally infect vertebrates. The baculovirus genome
includes the polyhedrin gene, whose product accumulates in the insect cell as large
nuclear inclusion bodies toward the end of the infection cycle (Figure 2.11). The
product of this single gene frequently makes up over 50% of the total cell protein.
Similar levels of protein production also occur if the normal gene is replaced by a
foreign one. Baculovirus vectors have been successfully used in production of a
number of mammalian proteins, but unfortunately the resulting proteins are not
glycosylated correctly. In this regard the baculovirus system does not offer any
advantages compared with S. cerevisiae or P. pastoris. However, the deficiencies in
the insect glycosylation process can be circumvented by using a modified baculovirus
that carries a mammalian promoter to express genes directly in mammalian cells.
The infection is not productive, meaning that the virus genome is unable to
replicate, but genes cloned into one of the BacMam vectors, as they are called, are
maintained stably in mammalian cells for enough time for expression to occur. This
expression is accompanied by the mammalian cell’s own posttranslational processing
activities, so the recombinant protein is correctly glycosylated and therefore should
be fully active.
Figure 2.11: Crystalline inclusion bodies in the nuclei of insect cells infected with a
baculovirus.
The Expression System Based on Bombyx mori Nucleopolyhedrovirus
Of course, in nature baculoviruses infect living insects, not cell cultures. For example,
one of the most popular baculoviruses used in cloning is the Bombyx mori
nucleopolyhedrovirus (BmNPV), which is a natural pathogen of the silkworm. There is
a huge conventional industry based on the culturing of silkworms for silk production,
and this expertise is now being harnessed for production of recombinant proteins,
29
using expression vectors based on the BmNPV genome. As well as being an easy and
cheap means of obtaining proteins, silkworms have the additional advantage of not
being infected by viruses that are pathogenic to humans. The possibility that
dangerous viruses are co-purified with the recombinant protein is therefore avoided.
2.9.3
Pharming—Recombinant Protein from Live Animals and
Plants
The use of silkworms for recombinant protein production is an example of the
process often referred to as pharming, where a transgenic organism acts as the
host for protein synthesis. Pharming is a recent and controversial innovation in gene
cloning.
2.9.3.1 Pharming in Animals
A transgenic animal is one that contains a cloned gene in all of its cells. Knockout
mice, used to study the function of human and other mammalian genes, are
examples of transgenic animals. With mice, a transgenic animal can be produced by
microinjection of the gene to be cloned into a fertilized egg cell. Although this
technique works well with mice, injection of fertilized cells is inefficient or impossible
with many other mammals, and generation of transgenic animals for recombinant
protein production usually involves a more sophisticated procedure called nuclear
transfer (Figure 2.12).
Figure 2.12: Transfer of the nucleus from a transgenic somatic cell to an oocyte.
30
This involves microinjection of the recombinant protein gene into a somatic cell,
which is a more efficient process than injection into a fertilized egg. Because the
somatic
cell
will
not
itself
differentiate
into
an
animal,
its
nucleus,
after
microinjection, must be transferred to an oocyte whose own nucleus has been
removed.
After implantation into a foster mother, the engineered cell retains the ability of the
original oocyte to divide and differentiate into an animal, one that will contain the
transgene in every cell. This is a lengthy procedure and transgenic animals are
therefore expensive to produce, but the technology is cost-effective because once a
transgenic animal has been made it can reproduce and pass its cloned gene to its
offspring according to standard Mendelian principles.
Although proteins have been produced in the blood of transgenic animals, and in the
eggs of transgenic chickens, the most successful approach has been to use farm
animals such as sheep or pigs, with the cloned gene attached to the promoter for the
animal’s -lactoglobulin gene. This promoter is active in the mammary tissue which
means that the recombinant protein is secreted in the milk (Figure 2.13).
Figure 2.13: Recombinant protein production in the milk of a transgenic sheep.
Milk production can be continuous during the animal’s adult life, resulting in a high
yield of the protein. For example, the average cow produces some 8000 liters of milk
per year, yielding 40–80 kg of protein. Because the protein is secreted, purification is
relatively easy. Most importantly, sheep and pigs are mammals and so human
31
proteins produced in this way are correctly modified. Production of pharmaceutical
proteins in farm animals therefore offers considerable promise for synthesis of
correctly modified human proteins for use in medicine.
2.9.3.2 Recombinant Proteins from Plants
Plants provide the final possibility for production of recombinant protein. Plants and
animals have similar protein processing activities, although there are slight
differences in the glycosylation pathways. Plant cell culture is a well established
technology that is already used in the commercial synthesis of natural plant
products. Alternatively, intact plants can be grown to a high density in fields. The
latter approach to recombinant protein production has been used with a variety of
crops, such as maize, tobacco, rice, and sugarcane. One possibility is to place the
transgene next to the promoter of a seed specific gene such as -phaseolin, which
codes for the main seed protein of the bean
Phaseolus vulgaris
The recombinant protein is therefore synthesized specifically in the seeds, which
naturally accumulate large quantities of proteins and are easy to harvest and to
process. Recombinant proteins have also been synthesized in leaves of tobacco and
alfalfa and the tubers of potatoes. In all of these cases, the protein has to be purified
from the complex biochemical mixture that is produced when the seeds, leaves, or
tubers are crushed. One way of avoiding this problem is to express the recombinant
protein as a fusion with a signal peptide that directs secretion of the protein by the
roots. Although this requires the plants to be grown in hydroponic systems rather
than in fields, the decrease in yield is at least partly offset by the low cost of
purification.
Whichever production system is used, plants offer a cheap and low-technology
means of mass production of recombinant proteins. A range of proteins have been
produced in experimental systems, including important pharmaceuticals such as
interleukins and antibodies. This is an area of intensive research at the present time,
with a number of plant biotechnology companies developing systems that have
reached or are nearing commercial production. One very promising possibility is that
plants could be used to synthesize vaccines, providing the basis to a cheap and
efficient vaccination program.
32
2.9.3.3 Ethical Concerns Raised by Pharming
With our discussion of pharming we have entered one of the areas of gene cloning
that causes concern among the public.
Transgenic Animals
With transgenic animals, one of the fears is that the procedures used might cause
suffering. These concerns do not center on the recombinant protein, but on the
manipulations that result in production of the transgenic animal. Animals produced
by nuclear transfer suffer a relatively high frequency of birth defects, and some of
those that survive do not synthesize the required protein adequately, meaning that
this type of pharming is accompanied by a high “wastage”. Even the healthy animals
appear to suffer from premature aging, as was illustrated most famously by “Dolly
the sheep” who, although not transgenic, was the first animal to be produced by
nuclear transfer. Most sheep of her breed live for up to 12 years, but Dolly developed
arthritis at the age of 5 and was put down one year later because she was found to
be suffering from terminal lung disease, which is normally found only in old sheep. It
has been speculated that this premature aging was related to the age of the somatic
cell whose nucleus gave rise to Dolly, as this cell came from a six-year-old sheep and
so Dolly was effectively six when she was born. Although the technology has moved
on dramatically since Dolly was born in 1997, the welfare issues regarding transgenic
animals have not been resolved, and the broader issues concerning the use of
nuclear transfer to “clone” animals (i.e., to produce identical offspring, rather than to
clone individual genes) remain at the forefront of public awareness.
Transgenic Plants
Pharming in plants raises a completely different set of ethical concerns, relating in
part to the impact that genetically manipulated crops might have on the
environment. These concerns apply to all GM crops, not just those used for
pharming.
References
1.
Gene Cloning and DNA Analysis: An Introduction, Sixth Edition. 2010. Brown TA. Blackwell
Science Ltd., Oxford.
2.
Genetics: Analysis of Genes and Genomes, Sixth Edition. 2005. Daniel L Hartl and Elizabeth W
Jones. Jones & Bartlett Publishers Inc., Boston.
3.
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition.
2010. Bernard R Glick, Jack J Pasternak and Cheryl L Patten. ASM Press, American Society for
Microbiology, Washington DC.
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