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UNIVERSITY OF NAIROBI
SCHOOL OF BIOLOGICAL SCIENCES
SBT 415 MOLECULAR BIOLOGY II
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.
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.
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.
∙ 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 β-
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 t ranscription and translation start signals
and control of expression by the lac r epressor.
∙ 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 a nd 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 w ill result.
∙ It will have a partial β-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.
IPTG, so addition of this chemical into the growth medium switches on transcription of a gene
inserted downstream of the lac p romoter carried by an expression vector.
romoter
(b) The trp p
∙ 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.
∙ 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 λ 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 λ OLPL operator/promoter
region.
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(b) The host cell used is a λ lysogen bearing a temperature-sensitive λ repressor gene ( cI8 57).
(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 (42oC), the temperature-sensitive
repressor is inactivated and removed from OL, allowing
transcription of the cloned gene to
occur.
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.
(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.
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 a re 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 a nd 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.
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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Human growth hormone Pituitary dwarfism Goeddel et al. (1979a) Insulin Diabetes
Goeddel et al. (1979b)
Tissue plasminogen activator Thrombosis (presence of blood blocking clot in vein
or consciousness)
artery) Pennica et al. (1983) Shine et al. (1980)
β-endorphin Analgesic (pain alleviating
without loss of
Interleukin 2 Cancer chemotherapy Taniguchi et al. (1983)
α1-antitrypsin Emphysema sacs)
(enlargement and lack of flexibility of Bollen et al. (1983)
air