TERM PAPER OF CONCEPTS IN

BIOTECHNOLOGY
( BTC – 012 )

TOPIC : EXPRESSION VECTORS AND THEIR

APPLICATIONS

SUBMITTED TO : SUBMITTED BY :
MR. HARSH KUMAR AYUSHI BANSAL
(LECTURAR IN BIOTECHNOLOGY) ROLL NO. - 02
1040070029
BTECH.BIOTECH
SEC-A
ACKNOWLEDGEMENT :

I would like to express my deep gratitude to my teacher “ MR. HARSH KUMAR ”

who gave me an opportunity to work on the topic “ EXPRESSION VECTORS AND
THEIR APPLICATION ” and also guided me a lot throughout the duration of this
term paper. I am also thankful to my parents and friends who constantly motivated
me and helped me a lot in the accomplishment of this term paper.

THANKING YOU ALL,

AYUSHI BANSAL
1040070029
CONTENTS :

• INTRODUCTION

• REVIEW OF LITERATURE

• EXPRESSION VECTORS

• EXPRESSION VECTORS USED FOR ANIMAL CELLS

• APPLICATIONS OF EXPRESSION VECTORS

• SITE DIRECTED MUTAGENESIS
• TRANSCRIPTION
• TRANSLATION
• PRODUCTION OF INSULIN
• DEVELOPMENT AND EXPRESSION OF siRNA EXPRESSION
VECTORS
• HETEROLOGOUS PROTEIN EXPRESSION IN BACILLUS SUBTILIS

• SUMMARY AND CONCLUSION

• BIBLIOGRAPHY
INTRODUCTION :

WHAT IS A VECTOR ?

A vector is a DNA molecule that has the ability to replicate autonomously in an

appropriate host cell and into which the DNA fragment to be cloned ( DNA insert ) is integrated
for cloning.

A vector must have following properties :

• IT SHOULD BE ABLE TO REPLICATE ANOMOUTOUSLY : When the objective of

cloning is to generate a large number of copies of the DNA insert , the vector replication
must be under relaxed control so that it can generate multiple copies of itself in a single
host cell.

• IT SHOULD BE IDEALLY LESS THAN 10 Kb IN SIZE : Because large DNA

molecules are broken during purification procedure. In addition , large vectors present
difficulties during various manipulations required for gene cloning.

• THE VECTOR SHOULD BE EASY TO ISOLATE AND PURIFY .

• IT SHOULD BE EASYILY INTRODUCED IN THE HOST CELLS , i.e. transformation

of the host cell with the vector should be easy.

• IT SHOULD HAVE SUITABLE MARKER GENES that allow easy detection of the
transformed host cells .

• IT SHOULD CONTAIN UNIQUE TARGET SITES for as many restriction enzymes as

possible into which the DNA insert can be integrated without disrupting an essential
function.

• WHEN EXPRESSION OF DNA INSERT IS DESIRED , the vector should contain at

least suitable control elements , e.g. promoters , operator and ribosome binding sites.

• WHEN THE OBJECTIVE IS GENE TRANSFER it should have the ability to integrate
either itself or the DNA insert it carries into the genome of the host cell.
Some important vectors used in hosts are :

1. CLONING VECTORS :

All vectors used for propagation of DNA inserts in a suitable host are called cloning
vectors.

PLASMID- A type of cloning vector

Illustration of a bacterium with plasmids enclosed showing chromosomal DNA and

plasmids.

A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal

DNA which is capable of replicating independently of the chromosomal DNA. In many
cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but
are sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in
Saccharomyces cerevisiae).

Plasmid size varies from 1 to over 200 kilobase pairs (kbp). The number of identical
plasmids within a single cell can range anywhere from one to even thousands under some
circumstances.

Plasmids can be considered to be independent life-forms similar to viruses, since both are
capable of autonomous replication in suitable (host) environments. However the plasmid-
host relationship tends to be more symbiotic than parasitic (although this can also occur
for viruses, for example with endoviruses) since plasmids can endow their hosts with
useful packages of DNA to assist mutual survival in times of severe stress. For example,
plasmids can convey antibiotic resistance to host bacteria, who may then survive along
with their life-saving guests who are carried along into future host generations.
There are two types of plasmid integration into a host bacteria: Non-integrating plasmids
replicate as with the top instance; whereas episomes , the lower example, integrate into
the host chromosome.

Types :
1. Conjugative plasmid :

Overview of Bacterial conjugation.

Conjugative plasmids contain so-called tra-genes, which perform the complex process of
conjugation, the transfer of plasmids to another bacterium (Fig. 4). Non-conjugative
plasmids are incapable of initiating conjugation,

Another way to classify plasmids is by function. There are five main classes:

• Fertility-F-plasmid, which contain tra-genes. They are capable of conjugation

(transfer of genetic material between bacteria which are touching).
• Resistance-(R)plasmids, which contain genes that can build a resistance against
antibiotics or poisons. Historically known as R-factors, before the nature of
plasmids was understood.
• Col-plasmids, which contain genes that code for (determine the production of)
bacteriocins, proteins that can kill other bacteria.
• Degradative plasmids, which enable the digestion of unusual substances, e.g.,
toluene or salicylic acid.
• Virulence plasmids, which turn the bacterium into a pathogen (one that causes
disease).

2. EXPRESSION VECTORS :

When a vector is designed for the expression of i.e. production of protein specified
by , the DNA insert , it is termed as expression vector. Such vectors contains atleast the
regulatory sequences , i.e., promoter , operator and ribosomal binding sites having
optimum function in the chosen host. It is desirable that all cloning vectors have relaxed
replication control so that they can produce multiple copies per host cell.

When an eukaryotic gene is to be expressed in a prokaryote , the eukaryotic coding

sequence has to be placed after prokaryotic promoter and a ribosome binding site , since
the regulatory sequences of eukaryotes are not recognised in prokaryotes. In addition ,
eukaryotic genes, as a rule , contain introns present within their coding regions. These
introns must be removed to enable the proper expression of eukaryotic genes since
prokaryotes lack the machinery needed for their removal from the RNA transcripts.
When eukaryotic genes are isolated as cDNA , they are intron – free and suitable for
expression in prokaryotes.
REVIEW OF LITERATURE :

Expression vector is a cloning vector that contains the necessary regulatory sequences to
allow transcription and translation of a cloned gene or genes and thus transcribe and
clone dna.
 Some strategies to construct the expression vectors.
 Expression vectors used for animal cells.
# pcDNA1.1/AMP
# Baculoviruses Vectors
# Alphavirus vectors
#Vaccinia Virus Vectors

 APPLICATIONS OF EXPRESSION VECTORS :

 Expression vectors are used for molecular biology techniques such as site-directed
mutagenesis. In general, DNA vectors that are used in many molecular biology gene
cloning experiments need not result in the expression of a protein. Expression vectors
are often specifically designed to contain regulatory sequences that act as enhancer
and promoter regions, and lead to efficient transcription of the gene that is carried on
the expression vector. Expression vectors are basic tools for biotechnology and the
production of proteins such as insulin that are important for medical treatments of
specific diseases like diabetes.

 Transcription

It includes 5 steps- Pre initiation, Initiation, Promoter clearance, Elongation,

Termination .

 Translation

* Basic mechanism

 Site-Directed Mutagenesis

 Production of Insulin

*Mechanism , production , regulation of production

 Application of siRNA expression vector

 Heterologous protein expression in Bacillus subtilis

Expression vector :
An expression vector, otherwise known as an expression construct, is generally a plasmid
that is used to introduce and express a specific gene into a target cell. Expression
vector allows production of large amounts of stable mRNA. Once the expression
vector is inside the cell, the protein that is encoded by the gene is produced by the
cellular transcription and translation machinery. The plasmid is engineered such that
it contains a highly active promoter which causes the production of large amounts of
mRNA.

After expression of the gene product, the purification of the protein is required; but since
the vector is introduced to a host cell, the protein of interest should be purified from the
proteins of the host cell. Therefore, to make purification process easy, the cloned gene
should have a tag. This tag could be histidine (His) tag or any other marker peptide .

Some strategies have been attempted for the construction of expression vectors
using regulatory sequences of the appropriate hosts. These are grouped in two
categories :

1. Construction of vectors allowing the synthesis of fusion proteins comprising amino

acids encoded by a sequence in the vector and those encoded by the DNA insert
(translational fusion) .

2. Development of vectors permitting the synthesis of pure proteins encoded exclusively

by the DNA inserts ( transcriptional fusion ).

Examples of the first strategy producing proteins are :

The expression of rat insulin , rat growth hormone , structural protein VP1 of foot and
mouth disease virus , human growth hormone etc.

Examples of second strategy producing unique proteins are :

Rabbit β-globin , small t- antigen of SV40 , human fibroblast interferon , human IGF-I
protein .The undesired amino acids encoded by the vector sequence in cases of
translational fusion must be removed from the fusion proteins by a suitable chemical
cleavage.
EXPRESSION VECTORS USED FOR ANIMAL CELLS :

1. pcDNA1.1/AMP :

This is highly versatile expression vector containing the following modules :

1. E . coli phage M13 origin ( generates single – stranded copies of the DNA insert )
2. CO1E1 origin ( for replication in E . coli )
3. SV40 origin ( for replication in COS cell lines )
4.Polyomavirus origin ( for replication in MOP-8 or WOP murine cell lines )
5. SV40 intron / polyadenylation site ( for enhanced transcription and polyadenylation of
mRNA )
6. A polylinker or multiple cloning site (MCS , for integration of transgene / DNA insert).
7. PCMV (human cytomegalovirus promoter for driving transgene transcription)
8. T7 promoter ( for in vitro transcription of the transgene )
9. Ampicillin resistance gene (for selection of recombinant E . coli clones) .

2. Baculoviruses Vectors :

Baculoviruses have double stranded DNA genomes. They productively infect

arthropods , particularly insects. These vectors are used mainly for high level transient
protein expression in insects and insect cells. The polyhedrin gene of baculoviruses can
be replaced by a transgene of interest , which is driven by the strong endogenous
polyhedrin promoter resulting in a high level of expression ( up to 1 mg / 106 cells ).

The main limitation of baculoviruses expression system is that glycosylation pathway of

insects differs from that of mammals. This problem can be resolved as follows:

• An insect cell line may be chosen for its ability to carry out mammalian type
glycosylations.

• The expression vector may carry a second transgene that encodes the specific
enzyme for the correct glycosylation of the recombinant protein.

• A transgenic cell line may be developed that expresses the appropriate enzyme
for glycosylation.

3. Alphavirus vectors :

Alphavirus like Sindbis virus and Semliki Forest virus ( SFV ) are enveloped viruses
having single – strand positive – sense RNA genome. Their replication occurs in the
cytoplasm producing a large number of genomes per cell ; therefore , the transgene
expression level is very high . The viral genome has only two genes :
1. A 5’- gene that encodes viral replicase.
2. A 3’- gene encoding a polyprotein.

Some of the strategies used to express recombinant proteins using alphavirus vectors are :

(i) Replication competent vectors are produced by inserting another subgenomic

promoter either upstream or downstream of the 3’- gene ; the transgene is inserted
downstream of this promoter. Such insertion vectors tend to be unstable and they have
been almost superceded by replacement vectors.

(ii) Replacement Vectors are produced by replacing the 3’- gene with the transgene. The
region of the first 40 codons of this gene of both Sindbis and SF viruses contain a strong
enhancer of protein synthesis. Therefore, this region is included in the recombinant DNA.
As a result , the recombinant protein is expressed as an N-terminal fusion protein. Such
vectors give very high yields ( up to 50% of total cellular protein ) of the recombinant
proteins.

4. Vaccinia Virus Vectors :

It is a close relative of the variola virus that causes small pox. The pox viruses have large
dsDNA genomes , which replicate in the host cell cytoplasm rather than the nucleus.
Therefore, they carry the entire DNA replication and transcription machinery in their
particles and the information for the same in their genomes. As a result the recombinant
genomes introduced into cells by transfection are non infectious.

Recombinant viruses are produced by homlogous recombination , using a targeting

plasmid transfected in to vaccinia virus infected cells. More recently , direct ligation
vectors have been developed ; these vectors are transfected into cells containing a helper
vaccinia virus to support replication and transcription of the recombinant DNA.

A binary expression system has been developed. In this system , one vaccinia vector
contains the transgene to be expressed under the control of strong T7 ( E . coli phage)
promoter. The other vaccinia vector contains the gene encoding T7 RNA polymerase
driven by a vaccinia virus promoter. Both the recombinant DNAs are used together to
transfect host cells. A cell transfected by both the recombinant DNAs produces T7
polymerase , which transcribes the T7 promoter - driven transgene ; this strategy
produces the recombinant protein up to 10% of the total cellular protein. Recombinant
vaccinia virus DNAs have been constructed to express a range of such important proteins
as HIV and HTLV III envelope proteins , hepatitis B surface antigen ( HbsAg ) .
APPLICATIONS OF EXPRESSION VECTORS :

Expression vectors are used for molecular biology techniques such as site-directed
mutagenesis. In general, DNA vectors that are used in many molecular biology gene
cloning experiments need not result in the expression of a protein. Expression vectors are
often specifically designed to contain regulatory sequences that act as enhancer and
promoter regions, and lead to efficient transcription of the gene that is carried on the
expression vector. Expression vectors are basic tools for biotechnology and the
production of proteins such as insulin that are important for medical treatments of
specific diseases like diabetes.

THE TWO MAIN APPLICATION OF EXPRESSION VECTORS LIES IN THE

PROCESSES OF TRANSCRIPTION AND TRANSLATION

1. SITE - DIRECTED MUTAGENESIS :

Site – directed mutagenesis is a molecular biology technique in which a mutation is

created at a defined site in a DNA molecule, usually a circular molecule known as a
plasmid. In general, site-directed mutagenesis requires that the wild-type gene sequence
be known.

This technique is also known as site-specific mutagenesis or Oligonucleotide-directed

mutagenesis.

2. TRANSCRIPTION :

Transcription is the synthesis of RNA under the direction of DNA. RNA synthesis, or
transcription, is the process of transcribing DNA nucleotide sequence information into
RNA sequence information. Both nucleic acid sequences use complementary language,
and the information is simply transcribed, or copied, from one molecule to the other.
DNA sequence is enzymatically copied by RNA polymerase to produce a complementary
nucleotide RNA strand, called messenger RNA (mRNA), because it carries a genetic
message from the DNA to the protein-synthesizing machinery of the cell. One significant
difference between RNA and DNA sequence is the presence of U, or uracil in RNA
instead of the T, or thymine of DNA. In the case of protein-encoding DNA, transcription
is the first step that usually leads to the expression of the genes, by the production of the
mRNA intermediate, which is a faithful transcript of the gene's protein-building
instruction. The stretch of DNA that is transcribed into an RNA molecule is called a
transcription unit. A transcription unit that is translated into protein contains sequences
that direct and regulate protein synthesis in addition to coding the sequence that is
translated into protein. The regulatory sequence that is before, or 5', of the coding

sequence is called 5' untranslated region (5'UTR), and sequence found following, or 3', of
the coding sequence is called 3' untranslated region (3'UTR). Transcription has some
proofreading mechanisms, but they are fewer and less effective than the controls for
copying DNA; therefore, transcription has a lower copying fidelity than DNA replication

As in DNA replication, RNA is synthesized in the 5' → 3' direction (from the point of
view of the growing RNA transcript). Only one of the two DNA strands is transcribed.
This strand is called the template strand, because it provides the template for ordering the
sequence of nucleotides in an RNA transcript. The other strand is called the coding
strand, because its sequence is the same as the newly created RNA transcript (except for
uracil being substituted for thymine). The DNA template strand is read 3' → 5' by RNA
polymerase and the new RNA strand is synthesized in the 5'→ 3' direction. RNA
polymerase binds to the 3' end of a gene (promoter) on the DNA template strand and
travels toward the 5' end.

Transcription is divided into 5 stages: pre-initiation, initiation, promoter clearance,

elongation and termination.

Pre-Initiation

Unlike DNA replication, transcription does not need a primer to start. RNA polymerase
simply binds to the DNA and, along with other cofactors, unwinds the DNA to create an
initiation bubble so that the RNA polymerase has access to the single-stranded DNA
template. However, RNA Polymerase does require a promoter like sequence. TATA
promoters are found around -10 and -35 bp to the start site of transcription.

Initiation

In bacteria, transcription begins with the binding of RNA polymerase to the promoter in
DNA. The RNA polymerase is a core enzyme consisting of five subunits: 2 α subunits, 1
β subunit, 1 β' subunit, and 1 ω subunit. At the start of initiation, the core enzyme is
associated with a sigma factor (number 70) that aids in finding the appropriate -35 and
-10 basepairs downstream of promoter sequences.

Transcription initiation is far more complex in eukaryotes, the main difference being that
eukaryotic polymerases do not directly recognize their core promoter sequences. In
eukaryotes, a collection of proteins called transcription factors mediate the binding of
RNA polymerase and the initiation of transcription. Only after certain transcription
factors are attached to the promoter does the RNA polymerase bind to it. The completed
assembly of transcription factors and RNA polymerase bind to the promoter, called
transcription initiation complex. Transcription in archaea is similar to transcription in
eukaryotes.Once the complex has been opened, initiation starts and the first
phosphodiester bond is formed. This is the end of initiation.

Promoter Clearance

After the first bond is synthesized the RNA polymerase must clear the promoter. During
this time there is a tendency to release the RNA transcript and produce truncated
transcripts. This is called abortive initiation and is common for both eukaryotes and
prokaroytes. Once the transcript reaches approximately 23 nucleotides it no longer slips
and elongation can occur. This is an ATP dependent process.

Elongation

One strand of DNA, the template strand (or non-coding strand), is used as a template for
RNA synthesis. As transcription proceeds, RNA polymerase traverses the template strand
and uses base pairing complementarity with the DNA template to create an RNA copy.
Although RNA polymerase traverses the template strand from 3' → 5', the coding (non-
template) strand is usually used as the reference point, so transcription is said to go from
5' → 3'. This produces an RNA molecule from 5' → 3', an exact copy of the coding strand
(except that thymines are replaced with uracils, and the nucleotides are composed of a
ribose (5-carbon) sugar where DNA has deoxyribose (one less oxygen atom) in its sugar-
phosphate backbone).

Unlike DNA replication, mRNA transcription can involve multiple RNA polymerases on
a single DNA template and multiple rounds of transcription (amplification of particular
mRNA), so many mRNA molecules can be produced from a single copy of a gene. This
step also involves a proofreading mechanism that can replace incorrectly incorporated
bases.

Prokaryotic elongation starts with the "abortive initiation cycle". During this cycle RNA
Polymerase will synthesize mRNA fragments 2-12 nucleotides long. This continues to
occur until the σ factor rearranges, which results in the transcription elongation complex
(which gives a 35 bp moving footprint). The σ factor is released before 80 nucleotides of
mRNA are synthesized.

In Eukaryotic transcription the polymerase can experience pauses. These pauses may be
intrinsic to the RNA polymerase or due to chromatin structure. Often the polymerase
pauses to allow appropriate RNA editing factors to bind.

Termination

Bacteria use two different strategies for transcription termination: in Rho- transcription
termination, RNA transcription stops when the newly synthesized RNA molecule forms a
G-C rich hairpin loop, followed by a run of U's, which makes it detach from the DNA
template. In the "Rho-dependent" type of termination, a protein factor called "Rho"
destabilizes the interaction between the template and the mRNA, thus releasing the newly
synthesized mRNA from the elongation complex. Transcription termination in eukaryotes
is less well understood. It involves cleavage of the new transcript, followed by template-
independent addition of As at its new 3' end, in a process called polyadenylation.

3. TRANSLATION :

Translation is the first stage of protein biosynthesis (part of the overall process of gene
expression). Translation is the production of proteins by decoding mRNA produced in
transcription. Translation occurs in the cytoplasm where the ribosomes are located.
Ribosomes are made of a small and large subunit which surrounds the mRNA. In
translation, messenger RNA (mRNA) is decoded to produce a specific polypeptide
according to the rules specified by the genetic code. This uses an mRNA sequence as a
template to guide the synthesis of a chain of amino acids that form a protein. Many types
of transcribed RNA, such as transfer RNA, ribosomal RNA, and small nuclear RNA are
not necessarily translated into an amino acid sequence. Translation proceeds in four
phases: activation, initiation, elongation and termination (all describing the growth of the
amino acid chain, or polypeptide that is the product of translation). Amino acids are
brought to ribosomes and assembled into proteins.

In activation, the correct amino acid is covalently bonded to the correct transfer RNA
(tRNA). While this is not technically a step in translation, it is required for translation to
proceed. The amino acid is joined by its carboxyl group to the 3' OH of the tRNA by an
ester bond. When the tRNA has an amino acid linked to it, it is termed "charged".
Initiation involves the small subunit of the ribosome binding to 5' end of mRNA with the
help of initiation factors (IF). Termination of the polypeptide happens when the A site of
the ribosome faces a stop codon (UAA, UAG, or UGA). When this happens, no tRNA
can recognize it, but a releasing factor can recognize nonsense codons and causes the
release of the polypeptide chain. The capacity of disabling or inhibiting translation in
protein biosynthesis is used by antibiotics such as: anisomycin, cycloheximide,
chloramphenicol, tetracycline, streptomycin, erythromycin, puromycin etc.

Basic mechanism
The mRNA carries genetic information encoded as a ribonucleotide sequence from the
chromosomes to the ribosomes. The ribonucleotides are "read" by translational
machinery in a sequence of nucleotide triplets called codons. Each of those triplets codes
for a specific amino acid.The ribosome and tRNA molecules translate this code to a
specific sequence of amino acids. The ribosome is a multisubunit structure containing
rRNA and proteins. It is the "factory" where amino acids are assembled into proteins.
tRNAs are small noncoding RNA chains (74-93 nucleotides) that transport amino acids to
the ribosome. tRNAs have a site for amino acid attachment, and a site called an
anticodon. The anticodon is an RNA triplet complementary to the mRNA triplet that
codes for their cargo amino acid.
Aminoacyl tRNA synthetase (an enzyme) catalyzes the bonding between specific tRNAs
and the amino acids that their anticodons sequences call for. The product of this reaction
is an aminoacyl-tRNA molecule. This aminoacyl-tRNA travels inside the ribosome,
where mRNA codons are matched through complementary base pairing to specific tRNA
anticodons. The amino acids that the tRNAs carry are then used to assemble a protein.
The energy required for translation of proteins is significant. For a protein containing n
amino acids, the number of high-energy Phosphate bonds required to translate it is 4n-1.
The rate of translation varies; it is significantly higher in prokaryotic cells (up 17-21
amino acid residues per second) than in eukaryotic cells (up to 6-7 amino acid residues
per second)

4. Development and application of siRNA expression vector

RNA interference (RNAi) is a sequence-specific silencing phenomenon, which is induced

by double-stranded RNA (dsRNA) and mediated through an evolutionary conserved
mechanism from plants to mammals. In mammalian cells, it has recently been reported
that 21- or 22-nucleotide (nt) RNAs with 2-nt 3' overhangs (siRNA) induce RNAi without
induction of the dsRNA-dependent inhibition of protein synthesis, known as the host
defense system against viral infections. Moreover, we and other have developed siRNA
expression systems utilizing a pol III promoter. Here we report a comparative analysis
among various siRNA expression vectors and also demonstrate a regulatable RNAi in
cells by using a tetracycline-controlled U6 promoter.

5. Design of an expression vector and its application to heterologous

protein expression in Bacillus subtilis :

An expression vector, pUBEX, was constructed for extracellular production of

heterologous proteins in Bacillus subtilis using a polyhistidine tag on the C-terminal
sequence, providing an efficient and easy purification of the protein. A CII protein, a
member of Bowman–Birk protease inhibitors, which was expressed as an inactive protein
in Escherichia coli, was successfully expressed in Bacillus subtilis using the pUBEX
vector and was purified to 6.4 mg l-1 by the immobilized metal affinity chromatography

6. Expression vectors also help in production of insulin.

Mechanism

Insulin is produced in the pancreas, and released when any of several stimuli are detected.
These include protein ingestion, and glucose in the blood (from food which produces
glucose when digested -- characteristically this is carbohydrate, though not all types
produce glucose and so an increase in blood glucose levels). In target cells, they initiate a
signal transduction which has the effect of increasing glucose uptake and storage. Finally,
insulin is degraded, terminating the response.

Production :
Insulin undergoes extensive posttranslational modification along the production pathway.
Production and secretion are largely independent; prepared insulin is stored awaiting
secretion. Both C-peptide and mature insulin are biologically active. Cell components
and proteins in this image are not to scale.

In mammals, insulin is synthesized in the pancreas within the beta cells (β-cells) of the
islets of Langerhans. One million to three million islets of Langerhans (pancreatic islets)
form the endocrine part of the pancreas, which is primarily an exocrine gland. The
endocrine portion only accounts for 2% of the total mass of the pancreas. Within the
islets of Langerhans, beta cells constitute 60–80% of all the cells.

In beta cells, insulin is synthesized from the proinsulin precursor molecule by the action
of proteolytic enzymes, known as prohormone convertases (PC1 and PC2), as well as the
exoprotease carboxypeptidase E. These modifications of proinsulin remove the center
portion of the molecule (ie, C-peptide), from the C- and N- terminal ends of proinsulin.
The remaining polypeptides (51 amino acids in total), the B- and A- chains, are bound
together by disulfide bonds/disulphide bonds. Confusingly, the primary sequence of
proinsulin goes in the order "B-C-A", since B and A chains were identified on the basis
of mass, and the C peptide was discovered after the others.

Regulation of production

The endogenous production of insulin is regulated in several steps along the synthesis
pathway:

• At transcription from the insulin gene.

• In mRNA stability.
• At the mRNA translation.
• In the posttranslational modifications.
SUMMARY :

An expression vector is generally a plasmid that is used to introduce and express a

specific gene into a target cell. Expression vector allows production of large amounts
of stable mRNA. Once the expression vector is inside the cell, the protein that is
encoded by the gene is produced by the cellular transcription and translation
machinery. The plasmid is engineered such that it contains a highly active promoter
which causes the production of large amounts of mRNA.

After expression of the gene product, the purification of the protein is required; but since
the vector is introduced to a host cell, the protein of interest should be purified from the
proteins of the host cell. Therefore, to make purification process easy, the cloned gene
should have a tag. This tag could be histidine (His) tag or any other marker peptide .

Expression vectors are used for molecular biology techniques such as site-directed
mutagenesis. In general, DNA vectors that are used in many molecular biology gene
cloning experiments need not result in the expression of a protein. Expression vectors are
often specifically designed to contain regulatory sequences that act as enhancer and
promoter regions, and lead to efficient transcription of the gene that is carried on the
expression vector. Expression vectors are basic tools for biotechnology and the
production of proteins such as insulin that are important for medical treatments of
specific diseases like diabetes.
BIBLIOGRAPHY :

The references used are :

Book referred :

BIOTECHNOLOGY EXPANDING HORIZONS

BY
B.D. SINGH

Pages – 36 to 38
330 to 336

Websites :

http://en.wikipedia.org/wiki/expression_vector
http://en.wikipedia.org/wiki/transcription_(genetics)
http://en.wikipedia.org/wiki/translation_(genetics)
http://en.wikipedia.org/wiki/site-directed_mutagenesis
http://en.wikipedia.org/wiki/insulin
http://en.wikipedia.org/wiki/vector_dna