Design Problem Title/No.

: II Course Code: BTY 375

Course Instructor: Nishtha mam Course Tutor (if applicable):

Date of Allotment: 29-01-2011 Date of submission: 21-02-2011

Student’s Roll No.: RA7801B22 Section No.: A7801

Declaration:
I declare that this assignment is my individual work. I have not copied from any other
student’s work or from any other source except where due acknowledgment is made
explicitly in the text, nor has any part been written for me by another person.

Student’s Signature: Nitin Thukral

Evaluator’scomments:
________________________________________________________________

Marks obtained: ___________ out of ______________________

Content of Homework should start from this page only:

Design an experiment to express a eukaryotic protein in a prokaryotic system.

The experiment should involve the following steps:

i) Selection of suitable vector (Justify the reason to prefer one type of vector over
the other)

ii) Extraction of foreign DNA

iii) Method to screen recombinant vector/ transformed host cell

iv) Steps to overcome the limitations associated with use of prokaryotic system

v) Method to check the expression and function of desired protein.

Solution:
The central dogma of molecular biology deals with the detailed residue-by-residue
transfer of sequential information. It states that information cannot be transferred back
from protein to either protein or nucleic acid.
Transcription of DNA to RNA to protein: This dogma forms the backbone of
molecular biology and is represented by four major stages.

1. The DNA replicates its information in a process that involves many enzymes:
replication.

2. The DNA codes for the production of messenger RNA (mRNA) during
transcription.

3. In eukaryotic cells, the mRNA is processed (essentially by splicing) and migrates

from the nucleus to the cytoplasm.

4. Messenger RNA carries coded information to ribosomes. The ribosomes "read" this
information and use it for protein synthesis. This process is called translation.

In this manner, a gene may be produced, which contains the. coding region from one
organism joined to regulatory sequences from another organism; such a gene is called
chimeric gene.
Recombinant DNA molecules are produced with one of the following three
objectives:
(1) to obtain a large number of copies of specific DNA fragments,

(2) to recover large quantities of the protein produced by the concerned gene, or

(3) to integrate the gene in question into the chromosome of a target organism where it
expresses itself. Even for the latter two objectives, it is essential to first obtain a large
number of copies of the concerned genes.

To achieve this, the DNA segments are integrated into a self-replicating DNA
molecule called vector; most commonly used vectors are either bacterial plasmids or
DNA viruses. This is achieved by using specific enzymes for cutting the DNA
(restriction enzymes) into suitable fragments and then for joining together the
appropriate fragments (ligation). In nature, gene transfers are rather imprecise, and
their range, in terms of species involved, is remarkably limited. The above problems
are circumvented by the recombinant DNA technology.

The entire procedure of cloning or recombinant DNA technology may be classified

into the following five steps for the convenience in description and on the basis of the
chief activity performed.

1. Identification and isolation of the desired gene or DNA fragment to be cloned.

2. Insertion of the isolated gene in a suitable vector.

3. Introduction of this vector into a suitable organism/cell called host

(transformation).

4. Selection of the transformed host cells.

5. Multiplication/expression/integration followed by expression of the introduced

gene in the host.

The protein Taken is Somatostatin (also known as growth hormone-inhibiting

hormone (GHIH) or somatotropin release-inhibiting factor (SRIF)) is a peptide
hormone that regulates the endocrine system and affects neurotransmission and cell
proliferation via interaction with G-protein-coupled somatostatin receptors and
inhibition of the release of numerous secondary hormones.
Somatostatin has two active forms produced by alternative cleavage of a single
preproprotein: one of 14 amino acids, the other of 28 amino acids.

In all vertebrates, there exists six different somatostatin genes which have been named
SS1, SS2, SS3, SS4, SS5, and SS6. Tetrapods only possess SS1 and SS2, while teleost
fish possess SS1 - SS6. The six different genes along with the five different
somatostatin receptors allows somatostatin to possess a large range of functions.

GENERAL PRCOEDURE

hGH (human growth hormone) used to be obtained from dead people's pituitary gland,
which was unsafe; then it started being 'manufactured' by genetic recombination.

Suppose you want to 'manufacture' a human protein in large amounts using bacteria.

1. First, take into account the differences between bacterial and eukaryotic molecular
processes. In particular, most human genes contain introns that must be spliced out of
the mRNA before it gets translated into a protein, but that process does not occur in
bacteria. So if we just put the human gene into bacteria, they will produce a defective
protein (one with all the pieces that should be taken out still left in).
So what we can do is isolate the mRNA and use reverse transcriptase to produce a
cDNA copy of the gene (since it is made from mature mRNA, the introns will have
already been spliced out). You can amplify the amount of cDNA using PCR, if
needed.

2. Then you create an expression vector. Suppose we use a bacterial plasmid.

a. Choose a plasmid that has resistance to one or more antibiotics so that we can
screen them.
b. Use a single restriction endonuclease (restriction enzyme) to digest both your
cDNA and the plasmid. Splice onto the end of your cDNA the sequences needed for
bacteria to transcribe and translate your gene. If we don't, we will amplify your DNA,
but it won't be expressed - it won't produce proteins in the bacteria. Then mix the two
sets of DNA fragments together with DNA ligase. Different combinations of DNA
will result: plasmid-plasmid, plasmid-cDNA, and cDNA-cDNA. You isolate the
recombinant DNA -- the plasmid-cDNA combinations.

3. Then introduce the recombinant plasmids into the bacteria. One method of doing
this is simply transformation.

4. You grow the bacteria on a medium that has the antibiotic that the plasmid provides
resistance to. That way, only the bacteria that have taken up your recombinant DNA
will survive.

5. The bacteria produce your protein along with others.

a. Vectors - A Vector is a DNA molecule that has the ability to replicate in an

appropriate host cell, and into which the DNA insert is integrated for cloning.
Therefore, a vector must have an origin of DNA replication (denoted as ori) that
functions in the host cell. Any extrachromosomal small genome, e.g., plasmid,
phage and virus, may be used as a vector.

pBR322 is a plasmid and for a time was one of the most commonly used E.
coli cloning vectors. Created in 1977, it was named eponymously after its
Mexican creators, p standing for plasmid, and BR for Bolivar and Rodriguez.

pBR322 is 4361 base pairs in length and contains a replicon region (source
plasmid pMB1), the ampR gene, encoding the ampicillin resistance protein
(source plasmid RSF2124) and the tetR gene, encoding the tetracycline
resistance protein (source plasmid pSC101). The plasmid has unique restriction
sites for more than forty restriction enzymes. 11 of these 40 sites lie within the
tetR gene. There are 2 sites for restriction enzymes HindIII and ClaI within the
promoter of the tetR gene. There are 6 key restriction sites inside the ampR gene.
The origin of replication or ori site in this plasmid is pMB1 (a close relative of
ColE1). The ori encodes two RNAs (RNAI and RNAII) and one protein (called
Rom or Rop).

The circular sequence is numbered such that 0 is the middle of the unique
EcoRI site and the count increases through the tet genes. The ampicillin
resistance gene is a penicillin beta-lactamase. Promoters P1 and P3 are for the
beta-lactamase gene. P3 is the natural promoter, and P1 is artificially created by
the ligation of two different DNA fragments to create pBR322. P2 is in the
same region as P1, but it is on the opposite strand and initiates transcription in
the direction of the tetracycline resistance gene. The molecular weight is 2.83 x
106 daltons.
b.

The isolated DNA is then treated with the suitable Restriction enzyme to extract the
foreign DNA.
The diagram below illustrates the DNA nucleotide sequence for the human
somatostatin gene insert. This diagram indicates where EcoRI and BamHI
each cut the human DNA sequence.

Recombinant somatostatin was made using EcoRI and BamHI to remove a

piece of the pBR322 plasmid. The human gene insert that was cut with these
same restriction enzymes was inserted into the plasmid. The diagram below
illustrates how the human somatostatin DNA and the pBR322 plasmid
DNA fit together using the “sticky” ends produced by these two enzymes.

• Human somatostatin gene in bold

• Plasmid DNA in not in bold
c. Screening

The pBR322 plasmid possesses genes coding for ampicillin and tetracycline
resistance. These gene markers facilitate the detection of bacteria that take up the
recombinant plasmid possessing the human gene for somatostatin. The transformed
bacteria are plated on agar containing one of the antibiotics. After growth,
replica plating is used to test the colonies for resistance to the other antibiotic.

a. The boxed colony represents a transformed colony of E. coli cells. Cells

that were not transformed with the recombinant plasmid are able to grow in
the presence of either antibiotic.
a. The boxed colony represents a transformed colony of E. coli cells. Cells that were
not
transformed with the recombinant plasmid are able to grow in the presence of either
antibiotic.
b. The restriction enzyme PstI would cut the ampicillin gene in the plasmid, rendering
it
inactive.
c. Therefore, cells that were transformed with the recombinant plasmid would be able
to grow
in the presence of tetracycline, but not in the presence of ampicillin.

a. The boxed colony represents a transformed colony of E. coli cells. Cells that were
not
transformed with the recombinant plasmid are able to grow in the presence of either
antibiotic.
b. Either of the restriction enzymes BamHI and Sal1 would cut the tetracycline gene in
the
plasmid, rendering it inactive.
c. Therefore, cells that were transformed with the recombinant plasmid would be able
to grow
in the presence of ampicillin, but not in the presence of tetracycline.
Prokaryotic Protein Expression Systems

Prokaryotic recombinant protein expression systems have several advantages. These

include ease of culture, and very rapid cell growth meaning you won't have to wait
long to get protein from bacterial systems once you clone your cDNA. Expression can
be induced easily in bacterial protein expression systems using IPTG. Also,
purification is quite simple in prokaryotic expression systems and there are a plethora
of commercial kits available for recombinant protein expression.

In the 1970s, when recombinant DNA technology found its way into molecular
biology laboratories, the bacterial cell was prevalently presented as a universal host
for heterologous protein expression. However, due to their inability to adequately
process complex proteins and due to their insufficient protein secretion capabilities
prokaryotic expression systems nowadays are mainly used for the production of rather
uncomplex proteins and peptides. In order to produce complex human recombinant
proteins a reinforced development of eukaryotic expression systems has proceeded in
the last decades, which was mainly based on yeast cells and mammalian cells. As a
result, mammalian cell-based biopharmaceuticals account for almost 60% of today's
biopharmaceutical market.

Also if you need to use your proteins for functional or enzymatic studies prokaryotic
systems are a problem as most proteins become insoluble in inclusion bodies and are
very difficult to recover as functional proteins. Furthermore, most if not all post-
translational modifications are not added by bacteria and therefore your protein of
interest may not be functional. Enzymatic studies thus may be unfruitful.
Various different expression systems which have been technologically used for
recombinant protein production were evaluated: an inducible prokaryotic expression
system (Escherichia coli) for intracellular human superoxide dismutase, an E. coli
secretion system for antibody Fab fragments, a constitutive eukaryotic secretion
system (Pichia pastoris) for human trypsinogen and antibody Fab fragments as well
as an intracellular P. pastoris system, and a constitutive eukaryotic expression system
(CHO) for an EPO/Fc fusion protein and human monoclonal antibodies. We
developed production scenarios for each expression system and compared specific
growth and productivity as well as product secretion rates to determine the full
potential in a bioprocess.

(e) When you want to characterise a gene or protein of interest, you must first study its
function. In this molecular era, obtaining a cDNA of your gene of interest is not
difficult.

To express the cDNA as a protein, ie. a recombinant protein, one can then easily
perform functional studies using the recombinant purified protein.

Once you have a purified protein you can conduct:

• Protein-protein Interaction Experiments

• Enzyme Kinetics
• Functional Studies of the Protein
• Structural Studies - including protein crystallization, protein structure and
NMR.
• You can also use the protein to make antibodies for further experiments.

There are two main systems for the expression of recombinant protein. Once you get
your cDNA cloned, you must decide where you want to amplify your protein. This
will be either a prokaryotic (bacterial) or eukaryotic (usually yeast or mammalian cell)
system. The choice of your system will decide which vector you will need to clone
your cDNA into as there are different promoters which function in E.Coli and others
that work best with yeast or mammalian systems.

The northern blot is a technique used in molecular biology research to study gene
expression by detection of RNA (or isolated mRNA) in a sample. A general blotting
procedure starts with extraction of total RNA from a homogenized tissue sample. The
mRNA can then be isolated through the use of oligo (dT) cellulose chromatography to
maintain only those RNAs with a poly(A) tail. RNA samples are then separated by gel
electrophoresis. Since the gels are fragile and the probes are unable to enter the
matrix, the RNA samples, now separated by size, are transferred to a nylon membrane
through a capillary or vacuum blotting system.

Hybridization is the technique in which the nucleic acid or protein immobilized on

the membrane is challenged with a probe or antibody known. Hybridization is widely
used to confirm the presence or absence of the DNA/RNA/protein in the unknown
sample. Hybridization depends on the function of the labelled base pair between
theProbe and the target sequences.

Gene expression levels are commonly determined using northern blot analysis.
However, this technique is time-consuming and requires a large quantity of RNA.
RT-PCR converts RNA into first strand cDNA, which is then used as a template for
PCR. RT-PCR is more rapid and sensitive and can be more specific than northern blot
analysis.

For this purpose mRNA molecules are first converted into cDNA sequences by means
of enzymic reactions involving the use of Reverse transcriptase. The resulting cDNAs
are then subjected to ordinary PCR amplification reactions. This technique is called
also mRNA phenotyping or Message amplification phenotyping since the use of
different primers specific for individual mRNAs can provide a snapshot picture of
gene expression within cells at a given time.

In all cases reaction mixtures obtained from amplification are subjected to gel
electrophoresis and amplified bands can then be visualized by appropriate staining of
the DNA.