Unit 1: Genetic engineering Compiled by Shanes

EXPRESSION VECTORS AND SYSTEM

INTRODUCTION TO EXPRESSION VECTORS AND THEIR IMPORTANCE:

Expression vectors are DNA molecules designed to facilitate the controlled expression of foreign genes
in host organisms. These vectors serve as delivery vehicles for inserting specific DNA sequences into
cells, allowing researchers to produce desired proteins. The ability to manipulate gene expression is
fundamental to genetic engineering and biotechnology, enabling applications ranging from basic
research to industrial production of pharmaceuticals and enzymes.

COMPONENTS OF EXPRESSION VECTORS:

Expression vectors consist of several key components, each contributing to the regulation and
optimization of gene expression:

• Promoter: The promoter region initiates transcription by providing a binding site for RNA
polymerase. It determines the strength and timing of gene expression.
• Coding Sequence (CDS): This is the segment of DNA that encodes the protein of interest. It is often
fused downstream of the promoter and upstream of other elements like ribosome binding sites
and terminators.
• Ribosome Binding Site (RBS): Also known as Shine-Dalgarno sequence in prokaryotes, RBS
facilitates the binding of ribosomes to the mRNA, ensuring efficient translation initiation.
• Terminator: The terminator sequence signals the end of transcription, preventing RNA
polymerase from continuing beyond the coding sequence. It ensures that the mRNA is correctly
processed.
• Selectable Marker: In many expression vectors, a selectable marker gene confers resistance to
specific antibiotics or enables the host cell to grow in media containing certain compounds. This
marker assists in identifying cells that have taken up the vector.
• Origin of Replication (ORI): ORI is necessary for replication of the vector within the host cells. It
ensures that the vector is faithfully propagated as the cells divide.
• Plasmid Backbone: The backbone of the vector provides the structural framework and regulatory
elements necessary for maintaining and replicating the vector in the host cell.

NEED FOR GENE CONSTRUCTS AND EXPRESSION SYSTEMS:

Gene constructs, created by fusing specific DNA sequences, enable researchers to precisely control the
expression of genes. These constructs are introduced into expression systems, which can vary from
prokaryotic (bacteria) to eukaryotic (yeast, mammalian cells) hosts. The choice of expression system
depends on factors such as the protein's complexity, post-translational modifications, and downstream
applications.

PROMOTERS:

Gene promoters are DNA sequences located upstream of gene coding regions and contain multiple cis-
acting elements, which are specific binding sites for protein involved in the initiation and regulation of
transcription. In most transcription units, the promoters are located next to transcription start site but
are not itself transcribed. Promoters normally contain a “core promoter”, which is a region around ~40bp

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upstream of the transcriptional initiation site which contains the TATA box. The TATA box is the binding
site for the transcription initiation factor TFIID TBP (TATA box binding protein) subunit.

Types of promoters used to regulate gene expression

There are different types of promoters used in genetic engineering. Promoters having its different
functions and role in gene regulation.

Figure: types of promoters used in gene construct and genetic engineering.

CONSTITUTIVE PROMOTERS

These promoters dictate expression in almost all tissues and are largely, if not entirely, independent of
environmental and developmental factors. As their expression is in general not conditioned by
endogenous factors. Constitutive promoters are usually active across species and even across kingdoms.

Plant pathogen promoters are commonly used as constitutive promoters: CAMV 35S and opine promoter,
Monocot promoters: Plant ubiquitin promoter (Ubi), Rice actin 1 (Act -1) and Maize dehydrogenase 1 (Adh
1).

Advantages of constitutive promoters:

• High level of production of proteins used to choose transgenic cells or plants

• High level of expression of reporter proteins or scorable markers, allowing easy detection and
quantification.
• High level of production of transcription factor that is part of a regulatory transcription system.
• Manufacturing of compounds that requires ubiquitous activity in the plants and
• Production of compounds that are required during all stages of plant development.

TISSUE SPECIFIC OR DEVELOPMENT STAGE SPECIFIC PROMOTERS:

Tissue specific promoters: which operate in particular tissues and at certain developmental stages of a
plant. They may be produced by endogenous and exogenous factors, so they may be also classified as
inducible. For plants promoter elements that are expressed or affect the expression of genes in the
vascular system such as photosynthetic tissues, tubers, roots, other vegetative organs, seeds,
reproductive organs can be found in heterologous systems. Tissue specific promoters are of three types:
root promoters, fruit promoters and seed promoters.

INDUCIBLE PROMOTERS:

These are only expressed under the presence of factors/ compounds because their expression is normally
limited to certain plant tissues. They can also be considered as tissue specific based on the nature of
factors that trigger their expression. They are classified into two groups:

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Chemically regulated: these are the chemical compounds, usually not naturally found within plants, switch
on promoter activity. Several of the types of promoters involve chimeric components gathered from
human, animal, fungal and bacterial sources. Pathogen related: ethylene, Salicylic acid, thiamine, Benzol.

Steroid regulated: Glucocorticoid receptors (GR) and glucocorticoid responsive element (GRE). Metal
regulated by copper, zinc, gold, cobalt etc.

SYNTHETIC PROMOTERS

Synthetic promoters are DNA sequences that do not found in nature and which are designed to regulate
the activity of genes controlling a gene’s ability to produce its own uniquely encoded protein.

RIBOSOME BINDING SITES (RBS):

RBS plays a crucial role in translation initiation. In prokaryotes, the Shine-Dalgarno sequence interacts
with the ribosome, positioning it correctly on the mRNA for efficient translation. Optimizing RBS
sequences can significantly enhance translation efficiency, improving the yield of the target protein.

TERMINATORS:

Terminator sequences mark the end of transcription. In bacteria, the rho-independent terminator forms
a stem-loop structure that causes RNA polymerase to detach from the DNA template. This ensures that
the transcribed mRNA is accurately processed and prevents read-through transcription.

REPORTER GENES:

Reporter genes are genes that encode easily detectable proteins or enzymes whose activity can be
monitored. These genes are often co-expressed with the gene of interest to provide insights into its
expression. For example, the gene encoding green fluorescent protein (GFP) can be used as a reporter to
visualize gene expression in real time.

FUSION PROTEINS AND TAGS

Expression of a gene and efficient translation of the expressed mRNA into protein is only the start of the
story when it comes to working with proteins. Once high level amounts of the protein have been produced
inside cells, the protein must then be purified before it can be studied in detail. This means that the protein
of interest has to be separated from all the other components of the cell, including all the other proteins.
Protein purification is a complex process requiring a high degree of skill and experience, and protocols for
purifying proteins often have to be laboriously devised and tested. Most protocols rely on finding
particular properties of the protein in question which can be used to gradually enrich the protein away
from all the other proteins and components in the cell. For example, proteins can be separated by size on
columns containing gels of the right sort, or by charge by binding them to a column and then gradually
washing them off in series of solutions of increasing salt concentration. Often, several of these procedures
have to be done one after the other until the protein obtained is highly pure.

More recently, a new range of techniques have come into general use which can speed up the process by
cutting down the number of steps necessary, and which are broadly applicable to a range of proteins with
very different properties. These techniques all involve adding extra amino acids to the protein that give it
a new property, which is to bind very tightly to a particular substance. This binding can then be used to

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isolate the protein away from all the other materials in the cell which do not bind this substance. The
method, called affinity chromatography, is best explained with a specific example.

To get the protein we need to allow the expression of our gene of interest (hence the name expression
vector) by employing the processes of transcription and translation.

Apart from the three DNA sequences discussed above (origin of replication, selectable markers and
multiple cloning sites), the expression vectors have some special additional sequences as well.

Those are as follows:

A. A bacterial promoter, such as the lac promoter. The promoter precedes a restriction site where
foreign DNA is to be inserted, allowing transcription of foreign sequence to be regulated by adding
substances that induce the pro-moter.
B. A DNA sequence that, when transcribed into RNA, produces a prokaryotic ribosome binding site.
C. Prokaryotic transcription initiation and termination sequences.
D. Sequences that control transcription initiation, such as regulator genes and operators.

In some types of expression vectors which are specifically used in association with the bacterial host (like
E. coli), multiple cloning site is not immediately adjacent to the ribo-some binding sequence, but instead
is preceded by a special sequence coding for a bacterial polypeptide.

While using such type of expression vectors the gene of interest is inserted just after the gene for bacterial
polypeptide. In this way we fuse two reading frames, producing a hybrid gene that starts with the bacterial
gene and progresses without a break into the codons of our gene of interest.

The product of gene expression is therefore a hybrid protein, consisting of short bacterial polypeptide
fused into amino terminus of our target polypeptide sequence. This hybrid polypeptide chain consist-ing
of two different types of polypeptides is called a fusion protein.

The followings are the reasons for incorporation of a fusion protein before our gene of interest:

(a) The presence of bacterial peptide at the start of fusion protein may stabilize the molecule and
prevent it from being degraded by the host cell. In contrast the for-eign polypeptides that lack a
bacterial seg-ment are often destroyed.
(b) The bacterial polypeptide may act as a sig-nal peptide, responsible for transporting our target
protein to a specific location from where these are collected. For example, if the bacterial peptides
are derived from a protein that is exported by the cell (e.g.; products of ompA genes), then our
target polypeptide will simply be transported outside of the host cell straight into the culture
media from where these can be collected.
(c) The bacterial polypeptide may also help in purification of the target polypeptide by different
purification techniques such as affinity chromatography.

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Figure: the key features of a typical expression vector

FUSION PROTEINS AND THEIR SIGNIFICANCE:

Fusion proteins merge distinct protein domains to create novel functionalities that can't be achieved with
individual proteins alone. They play a pivotal role in various biological applications, from basic research to
therapeutic development. By fusing proteins of interest, researchers can design experimental systems to
study protein-protein interactions, cellular localization, enzyme activities, and more. Fusion proteins
provide a unique window into the intricacies of cellular processes, shedding light on the underlying
molecular mechanisms.

AFFINITY TAGS AND THEIR TYPES:

Affinity tags are short peptide sequences strategically fused to a target protein to facilitate its purification,
detection, and characterization. These tags serve as handles for interactions with specific ligands or
matrices. Among the diverse range of affinity tags available, the His (histidine) and GST (glutathione-S-
transferase) tags have gained widespread popularity due to their efficiency and versatility.

• His Tags (Histidine Tags):

His tags consist of a series of histidine residues typically in a 6x configuration. The imidazole side
chains of histidines have a strong affinity for metal ions, particularly nickel and cobalt. This
property forms the basis for immobilizing His-tagged proteins on metal-chelating resins during
the purification process. His-tag purification is a cornerstone in protein biochemistry, allowing
researchers to isolate proteins with high specificity and purity.
• GST Tags (Glutathione-S-Transferase Tags):
GST tags are derived from the glutathione-S-transferase enzyme. These tags offer a high affinity
for glutathione, a tripeptide molecule. GST-tagged proteins can be purified using affinity
chromatography, where the fusion protein selectively binds to glutathione-coated beads or
matrices. The reversible interaction between GST tags and glutathione ensures efficient
purification while maintaining the native conformation of the tagged protein.

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PURIFICATION STRATEGIES FOR TAGGED PROTEINS:

The integration of affinity tags into fusion proteins streamlines the purification process. The following
steps outline the common procedure for purifying tagged proteins:

• Binding: The lysate containing the fusion protein is passed through a chromatography column or
beads containing the appropriate affinity matrix. The tag on the protein interacts specifically with
the immobilized ligand, leading to the target protein's attachment.
• Washing: Non-specific or weakly bound proteins and impurities are removed through a series of
washing steps. The tagged protein, securely bound to the affinity matrix, remains attached.
• Elution: By altering conditions such as pH or introducing competitive agents, the tagged protein
is released from the affinity matrix. The eluted protein is now in a purified state, ready for further
analysis or downstream applications.

Figure: Purification of protein tags

DETECTION OF TAGGED PROTEINS:

Tag detection is pivotal for confirming successful expression, assessing purification efficiency, and
enabling subsequent experimental analyses. A range of techniques can be employed to detect tagged
proteins:

• Western Blotting: Utilizing antibodies specific to the tag or the protein of interest, this method
enables the visualization of tagged proteins after gel electrophoresis.
• Immunoprecipitation: Tag-specific antibodies are used to isolate the tagged protein from a
complex mixture, allowing for detailed characterization.
• Mass Spectrometry: By measuring the mass-to-charge ratios of peptides, mass spectrometry
identifies proteins, including those carrying affinity tags, based on their unique mass signatures.
• Fluorescent Tags: Tags with intrinsic fluorescence properties can be directly visualized using
specialized imaging systems, eliminating the need for antibodies.

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Expression Systems: E. coli, Yeast, Insect Cells, Mammalian Cell Lines, and Cell-Free Extracts

Expression systems constitute the cornerstone of modern biotechnology, enabling the translation of
genetic information into functional proteins for a spectrum of scientific, therapeutic, and industrial
applications. The selection of an appropriate expression system depends on diverse factors such as the
complexity of the protein, the requirement for post-translational modifications, yield considerations, and
downstream objectives. This comprehensive exploration provides an in-depth analysis of prominent
expression systems including Escherichia coli (E. coli), yeast, insect cells, mammalian cell lines, and cell-
free extracts, shedding light on their distinct attributes, applications, and contributions to scientific
advancement.

E. coli Expression System:

E. coli, a prokaryotic microorganism, occupies a central role in genetic engineering due to its rapid growth,
established genetic background, and cost-effectiveness. Its application is most suitable for synthesizing
relatively straightforward proteins, as E. coli lacks the post-translational modification machinery
characteristic of eukaryotes. The E. coli system is particularly conducive to generating enzymes, peptides,
and proteins used in research, bioprocessing, and structural biology. Its remarkable yield and facile
manipulation render it ideal for high-throughput applications.

Yeast Expression System:

Yeast, exemplified by Saccharomyces cerevisiae, represents a eukaryotic expression system offering

distinct advantages. With its eukaryotic cellular machinery, yeast performs intricate protein folding and
post-translational modifications like glycosylation. This renders yeast conducive to producing therapeutic
proteins such as insulin. Notably, yeast's single-cell nature expedites genetic manipulation and enhances
yield. The yeast expression system spans research and industrial realms, serving as a platform for diverse
applications including functional studies and large-scale biopharmaceutical production.

Insect Cell Expression System:

Insect cell systems, epitomized by the Baculovirus-Insect Cell Expression System, bridge the gap between
unicellular organisms like yeast and complex mammalian cells. These systems are particularly well-suited
for proteins necessitating precise post-translational modifications like glycosylation and phosphorylation.
Insect cells, amenable to scale-up culture, offer an attractive avenue for producing viral proteins, vaccine
antigens, and recombinant proteins destined for structural analyses. This system's versatility has rendered
it pivotal in various domains of biotechnology.

Mammalian Cell Expression System:

Mammalian cell lines, including human cells, are unparalleled in their ability to produce proteins
demanding intricate post-translational modifications akin to those occurring in humans. This system
provides a biologically pertinent milieu, ensuring the generation of fully functional and correctly folded
proteins. In the realm of therapeutic protein production, mammalian cell expression assumes a critical
role, wherein protein conformation and modification are paramount for efficacy and safety. Its adoption
advances the production of biopharmaceuticals with desired pharmacokinetic and immunogenic profiles.

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CELL FREE SYSTEMS – RETICULOCYTE LYSATES

Rabbit reticulocyte lysate is prepared from the blood of animals that have been made anaemic
experimentally about 9–10 days before bleeding. Acetyl phenyl hydrazine injected daily for about four
days ensures anaemia, which, in turn, promotes an increased rate of red blood cell production in the
animal. The blood is collected in heparin solution (to prevent clotting) and the cells spun out of the fluid.
The cells are then lysed, centrifuged again, and the lysate stored at –70°C after freezing in liquid nitrogen.
The lysate is then treated with micrococcal nuclease (from Staphylococcus aureus).

The lysate is mixed with a haemin-containing (prevents haeme-directed inhibition of translation) buffer
that also has CaCl2. This mixture is incubated at 20°C with the nuclease. Reaction is stopped after 15–20
minutes by adding EGTA and the mixture kept on ice. This lysate can be stored in liquid nitrogen or used
right away. The enzyme-treated lysate is excellent for translating exogenous mRNAs, even when the latter
is present in very small quantities. The efficiency of the translation may, however, be lowered by the
continued presence of fragments of endogenous mRNAs containing the ribosome attachment (and,
therefore, translation initiation) site.

Figure: Comparison between in vivo system and CFPS (Cell free protein system)