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UNIVERSITY OF NAIROBI
SCHOOL OF BIOLOGICAL SCIENCES
SBT 415 MOLECULAR BIOLOGY II

Lecture 1: Expression vectors: production of recombinant protein

Specific objectives
By the end of this topic you should be able to:
1 Explain how an expression vector differs from a cloning vector.
2 List three elements that an expression vector should have for high-level inducible synthesis of
proteins.
3 Explain the nature of a fusion protein
4 Describe how gt 11 can be used to produce a fusion protein.
5 Explain briefly how positive gt 11clones in a cDNA library could be screened for expression of
a protein
6 Explain what an open reading frame is.
7 State the advantage of inducibility of an expression vector

Expression Vectors
 Any piece of DNA capable of autonomous replication within a host cell into which other DNA
sequences can be inserted and amplified is refereed to as a standard cloning vector.
 Unlike a standard cloning vector, an expression vector efficiently expresses an inserted foreign
gene by producing the protein product of the cloned gene, i.e. the recombinant protein.
 An expression vector should be propagated in its host cell as a single copy genomic insert to
enhance its stability, i.e. should occur only once in the host cell genome.
 It should also respond to induction with rapid increase in transcription of the foreign DNA.

Synthesis of a recombinant protein

 Depends on the presence of a collection of expression signals or short sequences of nucleotides
surrounding the foreign gene.
 These expression signals are a promoter, terminator and ribosome binding site
 The signals must be recognizable by E.coli and provide instructions for the transcriptional and
translational apparatus of the cell.
 Foreign gene is placed under the control of the E.coli expression signals.
 Genes of higher organisms are also surrounded by expression signals, but their nucleotide
sequences are not the same as the E. coli versions.
 Foreign gene is inactive in E.coli because the bacterium does not recognize its expression signals.
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1. Promoter.
 The promoter marks the point at which transcription of the gene should start.
 It is recognized by the sigma subunit of the transcribing enzyme RNA polymerase.
 A sigma factor is a protein component of prokaryotic RNA polymerases that binds loosely to the
core enzyme and restricts mRNA transcription to one of the two DNA strands and appropriate
promoter region.
 The promoter is the most important component of an expression vector because:
o It controls the first stage of gene expression i.e. the attachment of an RNA polymerase
enzyme to the DNA
o It determines the rate at which mRNA is synthesized.
o To some extent it determines the amount of recombinant protein
 A small variation has a major effect on the efficiency with which the promoter can direct
transcription.
 Most E.coli promoters do not differ much from the consensus sequence (TTGACA ), e.g.
TTTACA instead of TTGACA.
 Consensus sequences are conserved base sequences common in regulatory regions of DNA
e.g. promoter regions, where each base occurs in a particular position in a large proportion of
genomes of that species.
 Examples of consensus sequences are TATA box in eukaryotes (about 25-30 base pairs
upstream of the sequence to be transcribed) and Pribnow box sequences in prokaryotes
(TATAATG. also called a –10 region, because of the invariant T residue at base 10 upstream
from the start of the transcribed region).

One can distinguish between strong promoters and weak promoters.

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(a) Strong promoters (e.g. from phage) are those which can sustain a high rate of transcription.
They usually control genes whose translation products are required in large amounts by the cell.
(b) Weak promoters direct transcription of genes whose products are needed in only small
amounts. They are relatively inefficient.

2. Terminator
(a) It marks the point at the end of the gene where transcription should stop.
(b) It is usually a nucleotide sequence that can base-pair with itself to form a stem-loop structure.

3. Ribosome binding site (RBS)

(a) This is a short nucleotide sequence in mRNA molecule recognized by the ribosome as the point
for its attachment.
(b) RBS includes a Shine-Dalgarno sequence (SDS), which is a group of 6-8 purine-rich nucleotides
(5’ AAGGAGGU 3’) located in mRNA 4-15 bases upstream (5’) from the initiator codon AUG.
RBS is complementary to a region close to the 3’ end of 16S rRNA (the anti-SD sequence).

Factors to be consider when constructing an expression vector.

1. The expression vectors should carry a strong promoter, so that the cloned gene is transcribed
at the highest possible rate
2. It should have a strong translation initiation site.
3. It should be possible for the expression vector to regulate the promoter.
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There are two major types of gene regulation in E.coli, namely, induction and repression.
(a) Induction is the switching on of expression of a gene or group of genes in response to a chemical
or other stimulus. An inducible gene is one whose transcription is switched on by addition of a
chemical to the growth medium. Often this chemical is one of the substrates for the enzyme coded
by the inducible gene.

(b) Repression is the switching off of expression of a gene or group of genes in response to a chemical
or other stimulus. A repressible gene is switched off by the addition of the regulatory chemical.

Many of the sequences important in induction and repression lie in the region surrounding the
promoter and are therefore also present in an expression vector. It is therefore possible to extend the
regulation to the expression vector, so that the chemical that induces or represses the gene normally
controlled by the promoter is also able to regulate expression of the cloned gene.

The regulation of the cloned gene by the chemical that also induces or represses the gene
normally controlled by the promoter is an advantage in the production of recombinant protein
for two reasons.
1. If the recombinant protein has a harmful effect on the bacterium, then its synthesis must be
carefully monitored to prevent accumulation of toxic levels. This can be achieved by
judicious use of the regulatory chemical to control expression of the cloned gene.
2. Even if the recombinant protein has no harmful effects on the host cell, regulation of the
cloned gene is still desirable, as a continuously high level of transcription may affect the
ability of the recombinant plasmid to replicate, leading to its eventual loss from the culture.

Expression vectors permit the expression of the cloned sequences by fusing them to transcription
and translation start signals.

Expression vector that produces a fusion proteins e.g. gt 11

 gt 11 is a phage designed specifically as an expression vector.
 It contains the lac control region followed by the lacZ gene (the E.coli gene that encodes -
galactosidase)
 The cloning sites are located within the lacZ gene, so products of a gene inserted into this vector
will be fusion proteins with a leader of -galactosidase.
 The gene to be expressed is inserted into the EcoR1 site near the end of the lacZ coding region
just before the transcription terminator.
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 Upon induction of the lacZ gene by IPTG, a fused mRNA results, containing the inserted coding
region just downstream from that of the -galactosidase.
 This mRNA is translated by the host cell to yield a fusion protein.
 (A fusion protein is a protein resulting from the expression of a recombinant DNA containing two
open reading frames (ORFs) fused together. An open reading frame is a reading frame that is
uninterrupted by translation stop codons).

Detection of positive gt 11 clones

 gt 11 is suitable for making and screening cDNA libraries.
 It allows for direct screening of a group of clones for the expression of the right protein.
 The main components required for this procedure are a cDNA library in gt 11 and an
antiserum directed against the protein of interest.
 The  phages with various cDNAs are plated and the proteins released by each clone are blotted
onto a support such as nitrocellulose.
 The host cells lyse and in doing so release their products, making it easy to transfer proteins from
thousands of clones simultaneously, simply by touching a nitrocellulose filter to the surface of a
petri dish containing the plaques.
 Once the proteins from each plaque are transferred to nitrocellulose, they are probed with the
antiserum.
 Next, the antibody bound to protein from a particular plaque is probed by use of radioactive
protein A from Staphylococcus aureus.
 This protein binds tightly to antibody and makes the corresponding spot on the nitrocellulose
radioactive.
 The radioactivity is detected by autoradiography. The corresponding plaque is then picked from
the master plate.
Note
 A fusion protein is being detected and not the cloned protein by itself.
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 It does not matter if one has cloned a whole cDNA or not. The antiserum is a mixture of
antibodies that will react with several different parts of our protein, so even a partial gene will do,
as long as its coding region is cloned in the same orientation and reading frame as the leading -
galactosidase coding region.
 For instance, some vectors have restriction sites located just next to the control region for the lac
genes, which has been spliced into the vector. These restriction sites permit foreign DNA to be
spliced into the vector for cloning next to the lac control regions.
 This allows expression of the cloned gene by using the lac transcription and translation start
signals and control of expression by the lac repressor.
 Some vectors are bifunctional, allowing expression in two different hosts. For example, a certain
plasmid contain the origin of replication of the plasmid pBR322 and of the animal virus SV40.
 These origins allow replication in E.coli and some cultured mammalian cell lines,
respectively.
 These plasmids are called shuttle vectors, because they can transfer genes back and forth
from one type of cell to another.
 The pUC vectors place inserted DNA under the control of the lac promoter, which lies upstream
from the multiple cloning site.
 If an inserted DNA happens to be in the same reading frame as the lac gene it interrupts, a
fusion protein will result.
 It will have a partial -galactosidase protein sequence at its amino end and another protein
sequence, encoded in the inserted DNA, at its carboxyl end.

The foreign polypeptide can be recovered from the fusion protein by cleaving at the junction between
the two components with cyanogen bromide, which cuts polypeptides specifically at methionine
residues. The methionine residue at the fusion junction must be the only one present in the
entire polypeptide. If others are present then cyanogen bromide will cleave the fusion protein into
more than two fragments.

Promoters often used in expression vectors

(a) The lac promoter
This is the sequence that controls transcription of the lacZ gene coding for β-galactosidase (and also
the lacZ’ gene fragment carried by the pUC and M13mp vectors. The lac promoter is induced by
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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.

(b) The trp promoter

 The trp promoter 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 easily induced by 3-β-indoleacrylic acid.

(c) The tac promoter

This is hybrid between the trp and lac promoters that is stronger than either, but still induced by
IPTG.

(d) The λPL promoter

This is one of the promoters responsible for transcription of the λ DNA molecule. λPL is a very strong
promoter that is recognized by the E.coli RNA polymerase, which is subverted by λ into transcribing
the bacteriophage DNA. The promoter is repressed by the product of the λcl gene.
Expression vectors that carry the λP L promoter are used with a mutant E.coli host that synthesizes a
temperature-sensitive form of the cl protein. At low temperature (less than 30 oC) this mutant cl
protein is able to repress the λP L promoter. At higher temperatures the protein is inactivated resulting
in transcription of the cloned gene.

 It is usually advantageous to keep a cloned gene turned off until one is ready to express it.
There are three reasons for this:
(a) Eukaryotic proteins produced in large quantities in bacteria can be toxic.
(b) Even if the eukaryotic proteins are not toxic, they can build up to such great levels that they
interfere with bacterial growth.
(c) If the cloned were allowed to remain turned on constantly, the bacteria bearing the gene
would never grow to a great enough concentration to produce meaningful quantities of protein
product.

 The solution is keep the cloned gene turned off by placing it behind an inducible promoter that
can be turned off. One strategy is to use a very tightly controlled promoter such as the  phage
promoter PL.
 An example of a vector with such promoter is the pKC30.
(a) The gene to be expressed in inserted into the unique HpaI site of pKC30 vector, downstream
from the  OLPL operator/promoter region.
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(b) The host cell used is a  lysogen bearing a temperature-sensitive  repressor gene (cI857).
(c) When the temperature of these cells is kept relatively low (32oC), the repressor functions, and
no expression takes place.
(d) When the temperature is raised to the nonpermissive level (42 oC), the temperature-sensitive
repressor is inactivated and removed from O L, allowing transcription of the cloned gene to
occur.

Eukaryotic Expression Systems

Incompatibility of eukaryotic genes in prokaryotic cells

Eukaryotic genes are not compatible (not at home) in prokaryotic cells, even when they are expressed
under the control of the prokaryotic vectors. There are several reasons for this incompatibility.
1. E.coli cells frequently recognize the protein products of cloned eukaryotic genes as outsiders and
destroy them.
2. Prokaryotes don not carry out the same kinds of postranslational modification as do eukaryotes.
For example, a protein that would ordinarily be coupled to sugars in a eukaryotic cell will be
expressed as a bare protein when cloned in bacteria. This can affect a protein’s activity or
stability, or at least its response to antibodies.
3. The interior of a bacterial cell is not as conducive to proper protein folding as the interior of a
eukaryotic cell. Frequently, the result is improperly folded, inactive products of cloned genes.
4. A cloned gene can expressed at high level in bacteria, but the product forms highly insoluble,
inactive granules that are of no use unless one can somehow get the protein to dissolve and regain
it activity.
How to avoid incompatibility between a cloned gene and its host.
 This is done by expressing a gene in a eukaryotic cell.
 The initial cloning is done in E. coli, using a shuttle vector that can replicate in both bacterial and
eukaryotic cells.
 The recombinant DNA is then transferred to the eukaryote of choice by transformation. The
eukaryote commonly used for this purpose is yeast.
 There are several advantages of using yeast.
(a) Like bacteria, yeast grows rapidly and is easy to culture.
(b) By splicing (linking) the cloned gene to the coding region for yeast export signal peptide, one
ensures that the gene product will be secreted to the growth medium.
(A signal peptide is a stretch of about twenty amino acids, usually at the amino terminus of a
polypeptide, that helps to anchor the nascent polypeptide and its ribosome on the endoplasmic
reticulum. Polypeptides with a signal peptide are destined for packaging in the Golgi apparatus
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and are usually exported from the cell. The nascent polypeptide is extruded across the lipid
bilayer, where a signal peptidase cleaves the signal from the protein).
(c) Purification of the protein is easy. The yeast cells can be removed by centrifugation, leaving
relatively pure secreted gene product behind in the medium.

The yeast-based shuttle vectors

 They are based on the 2-micron plasmid that normally inhabits yeast cells.
 The plasmid provides the origin of replication needed by any vector that must replicate in yeast.
 The yeast-based shuttle vectors also contain the pBR322 origin of replication, so that they can
replicate in E.coli.
 They also contain a strong yeast promoter.
Another eukaryotic vector is derived from the nuclear polyhedrosis virus (NPV) that infects
the caterpillar known as the alfalfa looper. Viruses in this class have a rather large circular DNA
genome, approximately 130 kb in length. The major viral structural protein, polyhedrin, is made in
large quantities in infected cells. It has been estimated that when a caterpillar dies of a NPV infection,
up to 10% of the dry mass of the dead insect is this one protein. This indicates that the polyhedrin
gene must be very active apparently due to its powerful promoter.

General problems with the production of recombinant protein in E.coli

Although expression vectors have been developed, there are still many difficulties associated with the
production of protein from foreign genes cloned in E. coli. These problems can be grouped into those
that are due to:
(a) The sequence of the foreign gene, and
(b) Problems that stem from inherent properties of E. coli
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.
(a) The foreign gene might contain introns. This would be a major problem as E. coli genes do
not contain introns and the bacterium therefore does not possess the necessary machinery for
removing introns from transcripts.

(b) The foreign gene might contain sequences that act as termination signals in E. coli. These
sequences are perfectly innocuous in the normal host cell but in the bacterium result in
premature termination and loss of gene expression.
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(c) The codon usage 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 towards preferred codons.
This bias reflects the efficiency with which the tRNA molecules in the organisms are able to
recognize the different codons. If a cloned gene contains a high proportion of unfavoured
codons, then the host cell’s tRNA may encounter difficulties in translating the gene, reducing
the amount of protein that is synthesized.

Problems that stem from inherent properties of E. coli

1. 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 (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. This limits E. coli to
the synthesis of animal proteins that do not need to be glycosylated.
2. E. coli might not fold the recombinant protein correctly.
If the protein does not take up its correctly folded tertiary structure, it is usually insoluble and
forms an inclusion body within the bacterium. Recovery of the protein from the inclusion body is
not a problem, but converting the protein into the correctly folded form is difficult or impossible
in a test tube. Under these circumstances, the protein is inactive.
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3. E. coli might degrade the recombinant protein.

A fusion protein may be degraded less rapidly in E. coli than an unaltered recombinant protein.
These problems are less easy to solve than the sequence problems for the following reasons.
 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 be promoted by choosing a special host strain that
over-synthesizes the proteins thought to be responsible for protein folding in the cell.

Production of recombinant protein by eukaryotic cells

A microbial eukaryote such as 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 a batch or continuous culture systems, and may
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.

Two different systems for growth of microorganisms

(a) Batch culture system
A batch culture is one in which a culture of bacteria or microorganisms is in a fixed volume of liquid
medium in a closed vessel, with no additions or removals made during the period of incubation.

(b) Continuous culture system

A continuous culture is a culture of microorganisms in liquid medium under controlled conditions,
with additions to and removals from the medium over a lengthy period of time.
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Recombinant protein from fungi

Microbial eukaryotes are used for production of several animal proteins. Expression vectors are still
required because the promoters and other expression signals for animal genes do not in general work
efficiently in these lower eukaryotes.
Yeast
The yeast Saccharomyces cerevisiae is most frequently used microbial eukaryote for recombinant
protein synthesis for two reasons:
1. It is accepted as a safe organism for production of proteins for use in medicine or in foods
2. A wealth of knowledge regarding the biochemistry and genetics of S. cerevisiae exists. This
means it is relatively easy to devise strategies for minimizing the difficulties that arise from
its use.
Cloned genes are often placed under the control of the GAL promoter, which is normally upstream of
the gene coding for galactose epimerase, and enzyme involved in the metabolism of galactose. The
GAL promoter is induced by galactose, providing a system for regulating expression of a cloned
foreign gene.

Yields of recombinant protein are relatively high, but S. cerevisiae is unable to glycosylate animal
proteins correctly and lacks an efficient system for secreting proteins into the growth medium. Codon
bias can also be a problem. Despite these drawbacks, S. cerevisiae remains the most frequently used
microbial eukaryote for the reasons given above.

Another eukaryote used in recombinant protein synthesis is Pichia pastoris. It 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
exactly the same as the animal versions, but the differences are relatively trivial and would probably
not have a significant effect on the activity of a recombinant protein. In addition, the glycosylated
proteins made by P. pastoris are unlikely to induce an antigenic reaction if injected into the
bloodstream, a problem that is frequently encountered with the over-glycosylated proteins synthesized
by S. cerevisiae. Expression vectors for P. pastoris make use of the alcohol oxidase (AOX)
promoter, which is induced by methanol.

Filamentous fungi
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The two most popular filamentous fungi are Aspergillus nidulans and Trichoderma reesei (wood-rot
fungus). The advantages of these organisms are their
 good glycosylation properties and their
 ability to secrete proteins into the growth medium.
In its natural habitat Trichoderma reesei secretes cellulolytic enzymes that degrade the wood that it
lives on. The secretion characteristics mean that these fungi are able to produce recombinant protein
in a form that aids purification.
Expression vectors for Aspergillus nidulans usually carry the glucoamylase promoter, induced by
starch and repressed by xylose.

Expression vectors for Trichoderma reesei make use of the cellobiohydrolase promoter, which is
induced by cellulose.

Using animal cells for recombinant protein production

For protein with complex and essential glycosylation structures, an animal cell is the only type of
host within which the active protein can be synthesized.
Animal cells require a solid surface on which to grow. There are two ways of providing the solid
surface.
1. The vessel in which the cells are grown is filled inside with plates. This provides a large
surface area for growth. The disadvantage with this method is that complete and continuous
mixing of the medium within the vessel becomes very difficult.
2. A standard vessel is used but the cells are provided 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. However, this can be tolerated if it is the
only way of obtaining the active protein.

Although gene cloning may not be necessary in order to obtain animal protein, expression vectors and
cloned genes are used to maximize yield. This is achieved by placing the gene under control of a
promoter that is stronger than the one it is normally attached to.

A promoter that has been used in mammalian cells is the heat-shock promoter of the human hsp-70
gene, which is induced at temperatures above 40oC.

Another promoter used in mammalian cells is the mouse metallothionein gene promoter, which is
switched on by addition of zinc salts to the culture medium.
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Use of insect cells for animal protein production

Insect cells provide a potentially important alternative to mammalian cells for animal protein
production. Insect cells can provide high yields of recombinant protein. The expression system is
based on the baculoviruses. This is a group of viruses that are common in insects but apparently do
not infect vertebrates. The baculovirus genome includes the polyhedrin gene, whose normal product
accumulates in the insect cell as large nuclear inclusion bodies towards the end of the infection
cycle.
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. The insect cells in
many cases process proteins in the same way as mammalian cells but not always.

FOREING PROTEINS PRODUCED IN MICROORGANISMS

Protein Application Reference
Interferons Possible treatment of virus Houghton et al. (1980)
infections and cancer
Human growth hormone Pituitary dwarfism Goeddel et al. (1979a)
Insulin Diabetes Goeddel et al. (1979b)
Tissue plasminogen activator Thrombosis (presence of Pennica et al. (1983)
blood blocking clot in vein or
artery)
β-endorphin Analgesic (pain alleviating Shine et al. (1980)
without loss of
consciousness)
Interleukin 2 Cancer chemotherapy Taniguchi et al. (1983)
α1-antitrypsin Emphysema (enlargement Bollen et al. (1983)
and lack of flexibility of air
sacs)
Relaxin Facilitate childbirth Stewart et al. (1983)
Chymosin Cheese production Emtage et al. (1983)