2.
14 Recombinant Protein Expression and Purification
Fig. 2.15 RNAi method for gene
silencing. Dicer binds to dsRNA and
cleaves it to small fragments. The
fragments unwind and form RISC
complex. The RISC complex binds to
target mRNA and cleaves its sequence
specifically
target mRNA sequence specifically and degrades
it (Fig. 2.15).
MicroRNAs (miRNAs) are small noncoding
RNAs that are about 22 nucleotides long. They
mediate posttranscriptional silencing of target
genes. miRNAs are complementary to 3′ untrans-
lated region (UTR) of target genes and cause
silencing by translational inhibition, degradation
of mRNA, or both. They are synthesized in the
nucleus as pri-miRNA by RNA polymerase
II. Long pri-miRNAs are cleaved by Drosha-
DGCR8 to hairpin-like pre-miRNAs. These pre-
miRNAs are exported to the cytoplasm by
Exportin-5-Ran-GTP complex where they are
processed by Dicer enzyme to form short RNA
duplex. One strand of the duplex is incorporated
in RNA-induced silencing complex (RISC). The
miRNA loaded into RISC complex binds to the
target mRNA causing repression of translation or
mRNA degradation.
RNAi technique is widely used in biotechnol-
ogy to suppress gene expression. It can target and
silence any mRNA. It is used for library screen-
ing and is very useful to detect drug targets. This
technology has been successfully used to create
mutants. They are being developed as new class
of drugs.
43
Double stranded RNA (dsRNA)
Dicer binds to dsRNA
Dicer cuts dsRNA to siRNA
dsRNA unwinds and forms RISC
Binds to target mRNA and cleaves it
Degraded mRNA
2.14 Recombinant Protein
Expression and Purification
Recombinant protein expression and purification
refers to a set of techniques by which a protein of
interest is produced in a functional form in suffi-
cient quantity in a host organism by using host
protein synthesis machineries. The recombinant
protein expressed is purified to study its struc-
ture, functions, modifications, localization, and
interaction. In practical sense it involves cloning
the gene of interest in an expression vector and
expressing it in a suitable host that enables purifi-
cation of the recombinant protein in a functional
form in sufficient amount.
2.14.1 Protein Expression Systems
Both prokaryotic and eukaryotic systems are
used for protein expression depending on the
property of protein, the requirements for func-
tional activity, and yield. The important expres-
sion systems have been discussed below. The
advantages and disadvantages of each of them
have been tabulated in Table 2.1 (for further
details, refer to Chap. 4).
44 2 Fundamentals of Recombinant DNA Technology

Table 2.1 Recombinant protein expression system

Systems
Bacterial system
Yeast system
Insect cells with baculovirus
expression system
Cell-free system
Mammalian system
Merits
Fast growth rate
Simple growth requirements
Easy genetic manipulation
Fast growth rate
Simple growth requirements
Easy genetic manipulation
Posttranscriptional modification
Posttranslational modification
Large size of expression protein
Posttranscriptional modification
Posttranslational modification
Not restricted by host
Expression of toxic proteins
Suitable for mammalian protein
Expression of mAb
Problems
Posttranscriptional modification absent
Posttranslational modification absent
Protein folding options limited
Codon optimization required
Formation of inclusion bodies
Limited size
Codon optimization required
Expensive
Needs technical expertise
Expensive
Not suited for large-scale production
Expensive

2.14.1.1 Bacterial System
E. coli by far is the choice of organism for protein
purification as it has a fast growth rate with a dou-
bling time of 20 min [12]. It can grow to a very
high cell density which would increase expression
of recombinant protein. The growth requirements
are simple and inexpensive. Finally, there are a
large number of tools to genetically manipulate E.
coli and it is easily transformed [3].
The T7 system is the most popular approach
for producing proteins in E. coli. Here an expres-
sion vector containing a gene of interest cloned
downstream of the T7 promoter is introduced
into a T7 expression host. The host carries a chro-
mosomal copy of the phage T7 RNA polymerase
gene under inducible promoter. When inducer is
added, T7 RNA polymerase is expressed and
becomes dedicated to transcription of the gene of
interest. This mechanism has been discussed in
details in the promoters section below
(Sect. 2.14.2.4).
However, while expressing heterologous
genes in E. coli, it is important to consider codon
usage and optimization. All the mRNA codons
are not equally used in E. coli. The major codons
are those that occur in highly expressed proteins,
whereas the minor or rare codons tend to be in
genes expressed at a low level. Usually, the fre-
quency of the codon usage reflects the abundance
of their cognate t-RNAs. Therefore, when the
codon usage of the target protein differs signifi-
cantly from the average codon usage of the
expression host, this could cause problems dur-
ing expression. The problems due to different
codon usages are (1) decrease in mRNA stability
causing slowing down of protein expression; (2)
premature termination of transcription or transla-
tion producing truncated protein; (3) frameshift,
deletions, and mis-incorporation of amino acids;
and (4) inhibition of protein synthesis and growth
of cells. Codon optimization is replacing codons
that are rarely found in highly expressed E. coli
genes with more favorable codons throughout the
whole gene.
Despite several advantages, there are certain
limitations to protein expression in bacteria.
Multi-domain eukaryotic proteins expressed in
bacteria often are nonfunctional because the cells
are not equipped to accomplish the required
posttranslational modifications or molecular
folding. Also, many proteins become insoluble as
inclusion bodies that are very difficult to recover
without harsh denaturants and subsequent cum-
bersome protein-refolding procedures.
2.14 Recombinant Protein Expression and Purification
2.14.1.2 Fungal System
Expression of proteins in yeast is a common
alternative to prokaryotic and higher eukaryotic
expression. Yeast cells offer many of the
advantages of producing proteins in microbes
such as fast growth rate, easy genetic manipula-
tion, and simple growth conditions. It has some
of the properties of higher eukaryotic systems
such as posttranslational modifications and
secretory expression. Several yeast protein
expression systems exist in organisms such as
Saccharomyces, Pichia, Kluyveromyces,
Hansenula, and Yarrowia. The yeast expression
vectors contain yeast promoter, terminator, and
selectable marker. Many vectors have a secretory
sequence that secretes the expressed protein in
the medium. Most of these vectors can be main-
tained in E. coli as well and are known as shuttle
vectors. Thus, the gene of interest can be cloned
in E. coli and expressed in yeast. Some of the
yeast vectors can be integrated into the yeast
chromosome. They are stably maintained along
with the host chromosome.
2.14.1.3 Insect Cells and Baculovirus
Expression System
The baculovirus expression vector system
(BEVS) is one of the most powerful and versatile
eukaryotic expression systems available which
has been used to express heterologous genes
from different sources in insect cells.
Baculoviruses (family Baculoviridae) belong to a
diverse group of large double-stranded DNA
viruses that infect different species of insects as
their natural hosts. They are highly species spe-
cific and do not propagate in any non-invertebrate
host. The baculovirus genome is replicated and
transcribed in the nuclei of infected host cells
where the large baculovirus DNA (between 80
and 200 kb) is packaged into rod-shaped nucleo-
capsids. Since the size of these nucleocapsids is
flexible, recombinant baculovirus particles can
accommodate large amounts of foreign DNA.
In BEVS, several nonessential baculovirus
genes are replaced by heterologous genes. Since
the baculovirus genome is generally too large to
easily insert foreign genes, heterologous genes
are cloned into transfer vectors. Co-transfection
45
of the transfer vector and baculovirus DNA into
insect cell line, Spodoptera frugiperda (Sf),
allows recombination between homologous sites,
transferring the heterologous gene from the vec-
tor. Baculovirus infection of Sf cells results in the
shutoff of host gene expression allowing for a
high rate of recombinant mRNA and protein pro-
duction. Recombinant proteins can be produced
at levels ranging between 0.1 % and 50 % of the
total insect cell protein.
There are several advantages to use this system.
Functional activities of the recombinant proteins
are retained as insect cells are capable of post-
transcriptional (splicing) and posttranslational
modifications (phosphorylation, glycosylation,
and acylation). The size of expressed protein is
much larger. It is relatively a simple system.
2.14.1.4 Cell-Free System
Cell-free protein expression is performed without
the use of living cells. All the components needed
for transcription and translation such as ribo-
somes, t-RNAs, enzymes, cofactors, and amino
acids are provided in vitro. Such solutions are
obtained through making a cell lysate from a
desired cell type. Cell-free mixtures have been
made from both bacterial and eukaryotic cells.
However they are not useful for large-scale
protein expression. They are suited for a number
of applications where the rapid generation of a
smaller amount of recombinant protein is
desirable. Cell-free expression is suitable for
high-throughput screening of truncated proteins
for structural or functional studies. It is used to
express proteins that are toxic to expression hosts
in vivo. It is also used for expression of proteins
with modified amino acids, posttranslational
modifications, or studies on protein folding.
2.14.1.5 Mammalian System
In order to produce functional mammalian pro-
tein, sometimes it is required to express the gene
in mammalian cell line. The most widely used
host mammalian cells are Chinese hamster ovary
(CHO) cells, HEK293T, and mouse myeloma
cells, including NS0 and Sp2/0 cells. The vectors
used for expression in mammalian cell lines are
usually derived from mammalian viruses such as
46
adenovirus, vaccinia virus, retrovirus, and bacu-
lovirus. Mammalian cells are currently the main
hosts for commercial production of therapeutic
proteins, including monoclonal antibody (mAbs).
However, it is an expensive procedure and
requires technological skills [6].
2.14.2 Promoters
Several promoters are present that have their own
advantages and limitations with regards to heter-
ologous protein expression. Often combinations
of promoters and regulators are used to obtain a
favorable expression level. We will touch upon
the commonly used promoter systems for protein
expression.
2.14.2.1 Plac/PlacUV5
Most well-studied and commonly used promoter
in E. coli system is the lac promoter. It is the key
component of the lac operon and is induced by
lactose or its non-hydrolysable analog isopropyl
β-D-1-thiogalactopyranoside (IPTG). However,
it is repressed by the presence of glucose (Fig. 2.16)
by means of a process known as carbon catabo-
lite repression. In absence or low level of glu-
cose, cyclic adenosine monophosphate (cAMP)
is produced, which is necessary for complete
activation of the lac operon. To overcome catabo-
lite repression, a mutant lac promoter PlacUV5 is
lacI CAP Plac Operator lacZ
Binding
site
Fig. 2.16 The lac operon and its regulation. The lac
operon consists of lacZ, lacY, and lacA genes under the
control of lac promoter (Plac) and operator. It is suppressed
by lacI that binds to the operator sequences in presence of
glucose. In presence of lactose or IPTG, lacI is degraded
allowing the transcription of the operon by RNA poly-
merase. Under inducing condition, there is an increase in
cAMP and CAP protein that allows further upregulation
2 Fundamentals of Recombinant DNA Technology
used that has reduced sensitivity to catabolite
repression and is expressed in presence of glu-
cose (see Fig. 2.16). Lac promoter is negatively
regulated by LacI which is a suppressor of lac
operon. lacIQ is a mutation of lacI gene that pro-
duces very high levels of the lac repressor. This
provides a tight regulation of lac promoter and
stops leaky expression in the absence of inducer.
PlacUV5 in conjunction with lacIQ is present in several
expression vectors and provides stable expres-
sion in the presence of glucose and suppresses
leaky expression in the absence of inducer.
However, lac/lacUV5 promoters are weak.
2.14.2.2 tac/trc
The tac promoter is a synthetic hybrid promoter.
The promoter consists of the −35 region of the
trp (tryptophan) promoter and the −10 region of
the lac promoter. This is stronger than lacUV5
promoter and is used in pMAL series of vectors.
2.14.2.3 T7
T7 promoter system is extremely popular for pro-
tein expression and is present in pET vectors (for
figures and further details, refer to pET vectors in
Sect. 2.14.5.1). It requires T7 RNA polymerase
(T7 RNAP) that is expressed by λDE3 prophage
present in the host. Expression of T7 RNAP is
controlled by PlacUV5 promoter and is induced by
IPTG. Basal level of T7 RNAP expression is con-
trolled by lacIQ and also by the expression of T7
Glucose
lacY lacA
CAP protein
CAMP
RNA Polymerase
Lactose or IPTG
of the lac operon. PlacUV5 is a variant lac promoter that is
used in several expression vectors. RNAP is recruited to
this promoter more effectively resulting in higher rate of
transcription. Further, it works independently of activator
proteins and other cis regulatory elements other than the
basal promoter. LacIQ is a mutant LacI repressor protein
that tightly regulates Plac and PlacUV5 promoters
2.14 Recombinant Protein Expression and Purification 47

paraC pBAD
I1 I2
Genetic organization of arabinose
operon
araC O2 O1 CAP site araB araA araD

Repressor Operator Inducer Structural genes

O2

Arabinose absent
I1 I2

Arabinose present
O2 O1 I1 I2

CAP protein
CAMP

AraC

Fig. 2.17 Regulation of the arabinose operon. The arabi-
nose operon consists of three structural genes, araB, araA,
and araD. The araC gene encodes a transcriptional
repressor. In the absence of arabinose, the AraC repressor
protein binds the operator site, O2, and the inducer site I1,
causing the DNA in this region to adopt a looped confor-
mation preventing transcription. In the presence of arabi-
nose, the AraC repressor changes shape and binds the
adjacent inducer sites I1 and I2, causing the loop to be
released so that transcription may occur. Binding of the
CAP site by cAMP-CAP protein (CRP) leads to full
induction of expression

lysozyme expressed from a separate plasmid. T7
lysozyme binds to T7 RNAP and inhibits leaky
expression. After induction, excess T7 RNAP is
produced that are engaged in transcription of the
recombinant gene.
A hybrid T7/lac promoter has lacO operator
downstream of the T7 promoter to control basal
level of expression.
2.14.2.4 arapBAD
This expression system uses ara promoter of
arabinose operon and the dual repressor/activator
AraC protein (Fig. 2.17). In the absence of
arabinose inducer, AraC represses translation by
binding to two sites in the bacterial DNA. The
protein–DNA complex forms a loop, preventing
RNA polymerase from binding to the promoter.
Upon addition of the arabinose, AraC switches
into “activation mode” and promotes transcription
from the ara promoter. In this way, arabinose is
absolutely needed for induction.
2.14.2.5 pL Promoter
pL of phage lambda is expressed during early
lytic phase. It is tightly repressed by the λcI
repressor protein, which binds to the operator
sequences during lysogenic growth. When the
host SOS response is triggered by DNA damage,
the expression of the protein RecA is stimulated,
which in turn catalyzes the self-cleavage of λcI,
allowing transcription of pL-controlled genes
(Fig. 2.18). This mechanism is used in expression
vectors containing the pL promoter. Addition of
nalidixic acid, a DNA gyrase inhibitor, induces
SOS response and recombinant protein expres-
sion. Alternatively, λcI production can be con-
trolled by lac or trp promoters and can be induced
by IPTG or tryptophan, respectively. A mutant
48
a cI dimer
cI OR3 OR2 OR1
DNA damage causing RecA mediated cI degradation
cI OR3 OR2 OR1
PL GOI
cI/cI857
Fig. 2.18 (a) Regulation of lambda repressor (cI). The
life cycle of λ phage is controlled by cI and Cro proteins. It
remains in the lysogenic state when cI proteins predomi-
nate, but switches to lytic cycle when Cro proteins pre-
dominate. Transcription of the two proteins is regulated by
the cI protein and the operator sites. When cI dimer binds
to operators OR1 and OR2, Cro is repressed. When the host
DNA is damaged, RecA protease is expressed that cleaves
the cI protein. Cleaved cI proteins cannot bind to the oper-
ators. Thus, the Cro proteins are produced that transform
the λ phage into the lytic cycle. (b) Lambda repressor
λcI repressor protein (λcI857) is temperature
sensitive and is unstable at temperatures higher
than 37 °C. E. coli host strains containing the
λcI857 protein when shifted to higher tempera-
ture acts as an inducer.
2.14.3 Selection Markers
They are usually antibiotic-resistant genes that
stop the growth of E. coli that do not contain the
plasmid. Resistance to ampicillin is conferred by
the bla gene whose product β-lactamase is a peri-
plasmic enzyme that inactivates the β-lactam ring
of β-lactam antibiotics. However, it degrades the
ampicillin after sometime and then the cells
2 Fundamentals of Recombinant DNA Technology
λ genome lysogenic cycle
cro
λ genome lytic cycle
cro
Expression vector using lambda PL
Promoter and cI supressor
protein cI and operator sites are frequently used in expres-
sion vectors for regulating gene expression. The gene of
interest is cloned under the control of phage promoter (PL)
and operator sites. The gene encoding cI repressor is
expressed from the same construct and represses leaky
expression. When induced, the cI is degraded causing the
expression of the recombinant protein. λcI857 is tempera-
ture-sensitive cI repressor that is unstable at higher tem-
perature. The host strain is transferred to 42 °C for the
induction of gene expression. The cI and its derivatives
ensure stringent control over protein expression
lacking the plasmids can also grow in the
medium. Tetracycline has been shown to be
highly stable during cultivation, because resis-
tance is based on active efflux of the antibiotic
from resistant cells.
Antibiotics are expensive and are major causes
of development and spread of antibiotic resis-
tance. Hence alternative approaches such as plas-
mid addiction phenomenon are being used. Here
an essential gene is supplied by the plasmid that
is lacking in the host. Hence the host that loses
the plasmid is unable to survive. Different sub-
types of plasmid addiction systems exist such as
toxin-/antitoxin-based systems, metabolism-
based systems, and operator–repressor titration
systems. For example, dihydrofolate reductase
2.14 Recombinant Protein Expression and Purification
(DHFR) or glutamine synthase (GS) gene syn-
thesizes essential metabolite. In medium lacking
the metabolites, the transformed cells have
selective advantage due to presence of DHFR or
GS genes resulting in their growth.
2.14.4 Affinity Tags and Affinity
Purification
Affinity tags allow means for easy detection and
purification of recombinant protein from E. coli.
These are a stretch of amino acids (peptide tag)
or a large polypeptide (fusion partner) that are
expressed in tandem with the desired protein to
form a chimeric protein. Tags are used to increase
solubility of the protein as well. Small peptide
tags can form N-terminal or C-terminal fusions
and generally do not interfere with the function-
ing of the proteins. They are purified by using
affinity columns where the tagged protein binds
specifically and are later eluted. The common
peptide tags are the poly-Arg, FLAG, poly-His,
c-Myc, S, and Strep II tags. Commercial antibod-
ies are available for their detection by Western
blot analysis.
The 6xHis affinity tag is commonly used for
protein purification [11]. It is small, less immu-
nogenic, and uncharged at pH 8.0. It does not
generally affect secretion, compartmentalization,
or folding of the fusion protein within the cell. In
most cases, the 6xHis tag does not interfere with
the structure or function of the purified protein as
demonstrated for a wide variety of proteins,
including enzymes, transcription factors, and
vaccines. It allows the immobilization of the pro-
tein on metal-chelating surfaces and simplifies
many types of protein interaction studies. Anti-
His antibodies are available for detection by
Western blot or ELISA. C- or N-terminus fusion
with the protein of interest facilitates binding to
Ni2+ or Co2+ columns.
Non-peptide fusion partners increase solubility
of the protein of interest by working as a chaperon.
The most popular fusion tags are the maltose-
binding protein (MBP), N-utilization substance
protein A (NusA), and glutathione S-transferase
(GST). However the fusion partners are required
49
to be cleaved off eventually. Hence a cleavage
recognition site is usually incorporated in the
vector. In the case of tag removal by enzyme
digestion, expression vectors possess sequences
that encode for protease cleavage sites down-
stream of the gene coding for the tag. Some of the
commonly used cleavage sites are enterokinase,
thrombin, factor Xa, and the tobacco etch virus
(TEV) protease that have all been successfully
used for the removal of peptide tags and fusion
partners.
Fusion proteins with specific affinity tags sim-
plify their purification by employing affinity
chromatography methods. Immobilized-metal
affinity chromatography (IMAC) was first used
to purify proteins in 1975 by Porath et al. using
the chelating ligand iminodiacetic acid (IDA).
IDA was charged with metal ions such as Zn2+,
Cu2+, or Ni2+ and was used to purify a variety of
different proteins and peptides. However, IDA
has only three metal-chelating sites and cannot
bind metal ions tightly. This results in low yields,
impure products, and metal-ion contamination
of isolated proteins. Nitrilotriacetic acid (NTA)
column developed by QIAGEN for His-tagged
protein purification binds metal ions more stably
and retains the ions under a wide variety of
conditions, especially under stringent wash con-
ditions. NTA matrices can bind 6xHis-tagged
proteins more tightly than IDA matrices, allow-
ing one-step purification of proteins (Fig. 2.19).
The basic steps of IMAC have been explained in
Fig. 2.20. The His-tagged fusion protein is
expressed in E. coli. The cells are lysed to obtain
the protein either in native condition in the pres-
ence of phosphate or tris buffer or in denaturing
condition in the presence of urea or guanidine
HCl. The lysate is loaded on to the column for
binding. The imidazole rings in the histidine resi-
dues of the 6xHis tag bind to the nickel ions
immobilized by the NTA groups on the matrix.
Subsequently, the column is washed several
times with wash buffer containing low concentra-
tion of imidazole to remove impurities and non-
specific protein binding. Imidazole binds to the
nickel ions and disrupts the binding of histidine
residues in non-tagged background proteins. At
low imidazole concentrations, nonspecific, low-
50 2 Fundamentals of Recombinant DNA Technology

Fig. 2.19 Interaction of His

molecules with Ni2+ attached
to NTA matrix

Fig. 2.20 Affinity purification Expression vector

of His-tagged protein using
Ni-NTA column. The His Tagged protein
expression vector is induced in Cellular protein
the host. Recombinant protein
is expressed. The cells are
lysed and the lysate is loaded
on to Ni-NTA column for Cell lysis
binding. The column is washed
to remove cellular protein and
impurities. The tagged protein
is eluted in presence of high Binding to Ni-NTA column
concentration of imidazole.

Wash

Cellular protein in
wash through
Elution

Pure His-Tagged protein

2.14 Recombinant Protein Expression and Purification 51

affinity binding of background proteins is pre-
vented, while 6xHis-tagged proteins still bind
strongly to the matrix. Therefore, adding imidaz-
ole to the lysis buffer and wash buffer leads to
greater purity. The protein of interest is eluted
using high concentration of imidazole in the
elution buffer, which has structural resemblance
with histidine, and thus aids in elution of His-
tagged proteins (Fig. 2.21). 6xHis-tagged pro-
teins dissociate since they can no longer compete
for binding sites on the Ni-NTA resin.
2.14.5 Expression Vectors
Expression vectors have all the necessary ele-
ments for optimal gene expression. There are
several expression vectors available depending
NH3+ CH COO-
CH
N N
NH NH
Imidazole Histidine
Fig. 2.21 Structure of imidazole and histidine. Due to
similarity in structure, imidazole competes with histidine
for binding to the matrix. It is used for removal of nonspe-
cific binding and elution of His-tagged proteins during
affinity purification
on the host system, type of protein to be
expressed, and the nature of the experiment. The
important factors to consider while choosing a
vector have been tabulated in Table 2.2. We will
discuss essential features of E. coli expression
vectors.
Plasmid copy number of expression vectors is
an important consideration while choosing a
vector. It refers to number of plasmids that are
stably maintained in a host. It depends upon
the origin of replication of the plasmid. Usually
high copy number plasmids corelate with more
expression of the recombinant protein.
Commonly used expression vectors of pET
series use pMB1 origin and have 15–60 copies
per cell. pQE vectors from QIAGEN uses ColE1
origin and has 15–20 copies per cell. pBAD plas-
mids use p15A origin and are maintained at
10–12 copies per cell. However, if the protein is
toxic or imposes metabolic burden on the host, it
might cause a reduction in the growth rate and
exhibit plasmid instability or loss of plasmids.
Hence moderate to low copy number plasmid is
preferred for expression. The pSC101 is a low
copy number plasmid with less than five copies
per cell. It is used when high expression is
disastrous for cell growth. When more than one
plasmid types are used in a same host cell, they
should have different origins of replication;
otherwise, plasmid incompatibility would lead
to loss of one of the plasmids.
Expression vectors are equipped with strong
promoter, regulatory elements, appropriate initiation,

Table 2.2 Expression vector properties

Ori (copy number) Promoter Affinity tag Tag removal Selection marker
pMB101 (15–60) lac/lacUV5 Peptide affinity tags Thrombin Antibiotic resistance
ColE1 (15–20) tac/trc poly-Arg TEV Ampicillin
pUC (~500) T7 poly-His Factor Xa Kanamycin
p15A (10–12) arapBAD FLAG Enterokinase Tetracycline
pSC101 (3–5) c-Myc Chloramphenicol
Fusion partner Plasmid addiction system
MBP Toxin–antitoxin
NusA Metabolic markers
GST Operator–repressor
Ubiquitine
SUMO
52
Plasmids
Plasmid ColE1: ColE1 is a plasmid found in
bacteria. Its name derives as it carries a
gene for colicin E1 (the cea gene). It also
codes for immunity from this product
with the imm gene. In addition the plas-
mid has a series of mobility (mob) genes.
They are maintained in high copy number
in the cell and are used for cloning and
recombinant protein expression.
Commonly used plasmids derived from
ColE1 are pACYC, pUC18, pUC19,
pBluescript, pBR322, and derivatives [2].
Plasmid pMB1: pMB1 belongs to ColE1
family of plasmids and is maintained as
15–20 copies per cell. pMB1 origin is
used in cloning vector pBR322.
Plasmid p15A: Plasmid pl5A was first
detected as one of three plasmids in
Escherichia coli strain 15T. It is about
2.2 kb in size and one of the smallest
naturally occurring plasmids known. It
has been used to construct a cloning vec-
tor, pACYC184, which is useful because
it is readily maintained by bacteria also
carrying a ColEl derivative [15].
a b
PT7lac MCS T7 ter
pelB 6xHis
Ampr
lacI pET-22b
5493 bp
ori
Fig. 2.22 Schematic diagram of pET vectors. Transcription
is initiated by T7 promoter, regulated by lac operator/lacI,
and terminated by T7 terminator. They carry pBR322 ori-
gin of replication and multiple cloning sites (MCS). (a)
pET-22b carries N-terminal pelB signal sequence for peri-
2 Fundamentals of Recombinant DNA Technology
and termination sites and multiple cloning sites,
epitopes for protein detection by Western blot or
ELISA, and tags for affinity purification. Some
of the commonly used expression vectors are
described below.
2.14.5.1 pET
The pET vectors (Fig. 2.22) are developed for the
cloning and expression of recombinant proteins
in E. coli. The target genes cloned in pET plas-
mids are under the control of strong bacterio-
phage T7 transcription signal and are expressed
only when T7 RNA polymerase is provided by
the host cell. The T7 RNA polymerase is very
selective and active. Once induced, almost all of
the cell’s resources are converted to target gene
expression and the desired product can comprise
more than 50 % of the total cell protein.
The host E. coli strains are lysogen of bacte-
riophage DE3 and carry a DNA fragment
containing the lacI gene, the PlacUV5 promoter, and
the gene for T7 RNA polymerase (T7 RNAP).
The working of system is explained in Fig. 2.23.
This fragment is inserted into the int gene, pre-
venting DE3 from integrating into or excising
from the chromosome without a helper phage. In
a DE3 lysogen T7 RNAP gene is transcribed from
the lacUV5 promoter, which is inducible by
isopropyl-b-D-thiogalactopyranoside (IPTG).
Addition of IPTG to a growing culture of the lyso-
gen induces T7 RNAP, which in turn transcribes
6xHis
PT7lac MCS T7 ter
Thrombin 6xHis
Kanr
lacI pET-28a
5369 bp
ori
plasmic localization of the fusion protein and C-terminal
His-tag sequence for purification. This plasmid carries
ampicillin resistance marker. (b) pET-28a carry N-terminal
His tag, thrombin cleavage site, and optional C-terminal
His tag. This plasmid carries kanamycin resistance marker
2.14 Recombinant Protein Expression and Purification
a Host DE3
lacI IPTG
No inducer
Operator
lacUV5p RNAP gene
LacI binds to operator and inhibit expression
b PT7lac
GOI
Ampr
lacI pET vector IPTG
ori
Fig. 2.23 Molecular mechanism of pET vector expres-
sion in DE3 lysogen. (a) The host cell carries RNAP gene
under the control of PlacUV5 and lac operator. In absence of
IPTG, LacI suppressor binds to the operator sequences
and inhibits RNAP expression. In presence of IPTG, LacI
is degraded and RNAP gene is expressed. (b) The RNA
the target DNA cloned in the pET plasmid. In un-
induced state, the target genes remain transcrip-
tionally silent. Target genes are initially cloned
using hosts that do not contain the T7 RNAP
gene, thus eliminating plasmid instability due to
the production of proteins potentially toxic to the
host cell. Once established, the plasmids are
transferred into expression hosts and are induced
by the addition of IPTG. Two types of T7 pro-
moter (T7 and T7lac) and several hosts that differ
in their stringency of suppressing basal expres-
sion levels are available, providing great flexibil-
ity and the ability to optimize the expression of a
wide variety of target genes. The pET vectors are
designed to be able to read the target protein in all
the three reading frames and are designated with
suffix a, b, and c depending on the translational
start site with respect to the multiple cloning site.
53
lacUV5p RNAP gene
RNAP
LacI degradation and RNAP expression
PT7lac GOI
Ampr
lacI
ori
polymerase binds to the PT7lac promoter present in the pET
vector and induces transcription of the fusion protein
PT7lac. It is negatively regulated by LacI suppressor. In
absence of inducer, LacI binds to the operator sequences
and inhibits leaky expression of the fusion protein
They have highly efficient ribosome binding site
from the phage T7 major capsid protein. There are
several vectors in this family which differ in selec-
tion markers such as ampicillin and kanamycin
resistance. pET vectors also contain different
sequences adjacent to the cloning sites that encode
a number of peptide “tags,” which perform vari-
ous functions when fused with the target protein.
Some of the fusion tags facilitate detection and
purification of the target protein such as His tag,
S tag, GST tag, and T7 tag. Others increase the
probability of biological activity by affecting sol-
ubility in the cytoplasm (Trx tag) or export to the
periplasm (PelB/OmpT). N- and C-terminal His
tag and GST tags are mostly used for affinity puri-
fication. They carry protease cleavage sites such
as thrombin, enterokinase, and factor Xa peptide
cleavage site for removal of tags if required.
54
a EK
PBAD site MCS T7 ter
6xHis Epitope
Ampr
araC pBAD/His
A,B,C
4100 bp ori
Fig. 2.24 Schematic diagram of pBAD vectors.
Transcription is initiated by PBAD promoter, regulated by
AraC, and terminated by T7 terminator. They carry
pBR322 origin of replication, multiple cloning sites
2.14.5.2 pBAD
The pBAD expression vectors (Fig. 2.24) are
derived from low copy pBR322 plasmid and
are designed for regulated, dose-dependent
recombinant protein expression and purification
in E. coli. They utilize araBAD promoter (PBAD)
from E. coli for optimum levels of soluble,
recombinant protein expression. PBAD is turned
on in the presence of L-arabinose and turned off
in presence of glucose. Glucose reduces the lev-
els of 3′, 5′-cyclic AMP and cAMP activator pro-
tein (CAP) that is required for PBAD expression.
By varying the concentration of L-arabinose,
protein expression levels can be optimized to
ensure maximum expression of soluble protein.
The promoter is tightly regulated AraC which is
present in pBAD plasmid. AraC forms a complex
with arabinose and controls transcription. Hence
tight regulation of PBAD by AraC is useful for
expression of potentially toxic or essential genes.
There are two types of pBAD plasmids, pBAD/
His and pBAD/Myc-His. pBAD/His plasmid has
N-terminal polyhistidine tag for affinity purifica-
tion and act as epitope for antibody detection.
pBAD/Myc-His has C-terminal polyhistidine tag
and c-myc epitope. The epitopes are used for
detection of the protein by Western blot using
labeled secondary antibodies against them. The
tags and epitopes can be removed conveniently
by enterokinase digestion due to the presence of
enterokinase cleavage site. The pBAD plasmids
carry optimized ribosome binding site for
2 Fundamentals of Recombinant DNA Technology
b MCS
PBAD 6xHis T7 ter
myc
Ampr
araC pBAD/Myc-His
A,B,C
4100 bp ori
(MCS), and ampicillin resistance marker. (a) pBAD/His
carries N-terminal His-tag, epitope sequence followed by
enterokinase recognition site. (b) pBAD/Myc-His carries
myc epitope
FXa
PT5 site Stop
lacO 6xHis MCS
ColE1
Ampr pQE-30 Xa
3500 bp
Fig. 2.25 Schematic diagram of pQE vector. It carries
phage T5 promoter followed by two lac operator
sequences for controlled gene expression. It has
N-terminal His tag followed by factor Xa protease recog-
nition sequence. It has two strong transcriptional termina-
tors. pQE carries ColE1 origin of replication and
ampicillin resistance gene
increased efficiency of recombinant fusion pro-
tein expression, initiation ATG for translational
initiation, multiple cloning sites for insertion of
gene of interest, rrnB transcription termination
region for efficient transcription termination, and
ampicillin resistance gene (β-lactamase) for
selection of the plasmid in E. coli. pBAD plas-
mids are available as A, B, and C for expressing
the fusion protein in all three reading frames.
2.14.5.3 pQE
pQE vectors are shown in Fig. 2.25. The vectors
from QIAGEN comprise a family of vectors that
is used for expression of 6xHis-tagged recombi-
nant proteins in bacterial, baculovirus, and mam-
2.15 Chapter End Summary
malian expression systems. The pQE vectors
contain an optimized promoter–operator element
consisting of phage T5 promoter that is recog-
nized by E. coli RNA polymerase and two lac
operator sequences which increase lac repressor
binding and ensure efficient repression of the
powerful T5 promoter when not induced. They
carry synthetic ribosomal binding site, RBSII, for
high translation rates, 6xHis-tag coding sequence
either 5′ or 3′ to the cloning region, multiple
cloning site, and translational stop codons in all
reading frames. The pQE vectors have two strong
transcriptional terminators: t0 from phage lambda
and T1 from the rrnB operon of E. coli, to pre-
vent read-through transcription and ensure stabil-
ity of the expression construct. They have
β-lactamase gene (bla) conferring resistance to
ampicillin and ColE1 origin of replication. The
6X His tag can be added to N-terminal or
C-terminal end of the protein. The fusion protein
tagged with six consecutive histidine residues
can bind to nickel-nitrilotriacetic acid (Ni-NTA)
metal-affinity chromatography matrices and can
be affinity purified.
2.14.5.4 pGEX
pGEX vectors are shown in Fig. 2.26. They are
used for inducible, high-level expression and
purification of recombinant fusion protein with
glutathione S-transferase (GST) gene of
Schistosoma japonicum. Expression in E. coli
yields fusion proteins with the GST moiety at the
Ptac MCS T7 ter
GST Protease
Recognition
Site Ampr
pGEX
lacIQ
4900 bp
pBR322 ori
Fig. 2.26 Schematic diagram of pGEX vector. It carries
Ptac promoter followed by glutathione S-transferase and
protease recognition sequence. N-terminal GST fusion
protein is expressed. pGEX carries pBR322 origin of rep-
lication and ampicillin resistance gene
55
amino terminus and the protein of interest at the
carboxyl terminus. Expression is under the con-
trol of the tac promoter, which is induced by the
lactose analog isopropyl β-D thiogalactoside
(IPTG). The pGEX vectors carry lacIq gene. The
lacIq gene product is a repressor protein that
binds to the operator region of the tac promoter,
preventing expression until induction by IPTG,
thus maintaining tight control over expression of
the insert. The fusion protein accumulates within
the cytoplasm. GST fusion proteins are purified
from bacterial lysates by affinity chromatography
using immobilized glutathione. Glutathione is
attached to Sepharose and the structure of gluta-
thione is complementary to the glutathione
S-transferase binding site. GST fusion proteins
are captured by the affinity medium, and impuri-
ties are removed by washing. Fusion proteins are
eluted under mild, non-denaturing conditions
using reduced glutathione. The purification pro-
cess preserves protein antigenicity and function.
If desired, cleavage of the protein from GST can
be achieved using a site-specific protease such as
thrombin or factor Xa, whose recognition
sequence is located immediately upstream from
the multiple cloning site on the pGEX plasmid.
The pGEX vector series consists of pGEX-P,
pGEX-T, and pGEX-X that provide all three
translational reading frames.
2.15 Chapter End Summary
• Molecular cloning refers to a set of experi-
ments that is used to construct a recombinant
DNA molecule that can be replicate it in a
host. It includes amplification of the gene of
interest by PCR, restriction digestion of the
insert and the vector, ligation of the fragments,
and transformation into an appropriate host.
Several kinds of cloning vectors are available
depending upon the fragment size and the
need of the experiment. They are plasmid,
bacteriophage, cosmid, BAC, YAC, and HAC.
Bacterial host and plasmid systems are by far
the most popular. The bacterial lac operon is
modified and used for selection of clones