Cloned Genes and

Production of Growth
Hormones, Vaccines and
Commercial Chemicals
Human Peptide Hormone
Genes
In the human body, peptide
hormones are secreted after
encoding by peptide hormone genes
in the specialized cells, for instance
insulin and other Human Growth
Hormones (hGH).
Insulin
This peptide hormone i.e. insulin is
secreted by the Islets of Langerhans
of pancreas which catabolizes
glucose in blood. Insulin is a boon
for the diabetics whose normal
function for sugar metabolism
generally fails.
Insulin consists of two polypeptide
chains, chain A (21 amino acid long)
and B (30 amino acid long). Its
precursor is proinsulin which also
contains two polypeptide chains, A
and B, and is connected with a third
peptide chain-C (35 amino acid
long). However, the recent
discoveries reveal that precursor of
insulin is pre- pro insulin which is
about 109 amino acids long. The
pre-pro- insulin is synthesized in
beta cells of pancreas, the structure
of which is given below :
NH
2
-(Peptide)--Chain- (peptide-c)-
A chain-COOH
In the beginning, efforts were made
to isolate mRNA for pre- pro- insulin
from rats Islets of Langerhans of
pancreas and to synthesize cDNA.
Thereafter, it was inserted into a
plasmid. The recombinant plasmids
were transferred into the E. coli cells
which secreted pro-insulin.

Content
Cloned genes and production of
chemicals
Human peptide hormone genes
Insulines
Somatotropin
Somatostatin
-endorphin
Human interferon genes
Genes for vaccines
Vaccine for hepatitis-B virus
Vaccines for Rabies virus
Vaccines for poliovirus
Vaccine for foot and mouth
disease virus
Vaccines for small pox virus
Malaria vaccines
DNA vaccines
Genes associated with genetic
diseases
Phenylketonuria
Urokinase
Thalassaemia
Hemophilia
Enzyme engineering
Commercial chemicals
Prevention, diagnosis and cure of
diseases
Prevention of diseases
Diagnosis of diseases
Parasitic diseases
Monoclonal antibodies
Antenatal diagnosis
Gene therapy
Types of gene therapy
Methods of gene therapy
Success of gene therapy
Potential of gene delivering
system
Future needs of gene therapy
in India
DNA profiling (fingerprinting)
Methods of DNA profiling
Application of DNA profiling
Genetic databank
Reuniting the lost children
Solving disputed problems of
parentage, identity of criminals,
rapists, etc
Immigrant dispute
Hurdles of DNA profiling




Fig, 5.2. Production of recombinant
insulin in E.coli.


Itakura et. al. (1977) chemically
synthesized DNA sequence for two
chains, A and B, of insulin and
separately inserted into two pBR322
plasmids by the side of -
galactosidase gene. The
recombinant plasmids were
separately transferred into E
coli cells which secreted fused -
galactosidase - A chain and -
galactosidase - B chain separately.
These chains were isolated by
detaching from -glactosidase in
pure form in a amount of about 10
mg/24 g of healthy and transformed
cells (Sasson, 1984). Production of
recombinant insulin is shown in Fig.
5.2.
Animal and plant improvement
Transgenic Farm Animals
Crop Improvements
Transgenic plants
Nif gene transfer
Phaseolin gene transfer
Conversion of C
3
plants to
C
4
plants
Herbicide resistant plants
Insect pest resistant plants
Plant improvement through
genetic transformation
Crop Protection
Use of antagonists
Use of insecticides
Abatement of pollution

Detachment of proinsulin could
be possible when an extra
methionine codon was added at
the N-terminus of each gene for
A and B chains. The two chains
(A and B) were joined in
vitro to reconstitute the native
insulin by sulphonating the two
peptides with sodium
disulphonate and sodium
sulphite. Gilbert and
Villakomaroff (1980) isolated
mRNA for insulin from B cells of
rat's pancreas and inserted into
pBR322 plasmid in the middle
of a gene normally coding the
penicillinase, and incorporated
it into E. coli cells. E. coli cells
produced a hybrid protein
(penicillinase+proinsulin) from
which the proinsulin was
separated by using trypsin. It is
estimated that clones of E.
coli are capable of producing
about one million molecules of
insulin per bacterial cell. Human
insulin (humulin) is the first
therapeutic product produced
by means of recombinant
technology by Eli Lilly & Co.
Somatotropin
Somatotropin, the hGH, is
secreted by the anterior lobe of
pituitary glands which consists
of 191 amino acid units. Its
secretion is regulated by two
other hormones (somatostatin
and growth hormone releasing
hormone) produced by
hypothalamus. Deficiency of
somatotropin in about 3%
cases is of hereditary. It has
been estimated to about 1 child
in 5,000.



Fig. 5.3. Expression of Human Growth Hormone
(hGH) in E.coli; Lac P/O, lactose
promoter/operator.
Turner's syndrome is one of the most common chromosome disorders in girls
and it is characterized by short stature and non-functioning of ovaries affecting
approximately 1 in 2,500 live female birth. The extraction of somatotropin
pharmaceutically from the pituitary glands could not meet annual demand of this
hormone. Biosynthesis of somatotropin was achieved through gene cloning
procedures.

Double stranded cDNAs were produced from mRNA precursor of hGH which was
then incorporated into bacterial cells where it expressed in non-precursor form.
Bacteria were unable to convert peptide into biologically active form. A
recombinant plasmid containing a full length hGH cDNA (which fails to express)
is cleaved with restriction enzymes that release a fragment containing the
complete hGH coding sequence after codon 24 (Fig. 5.3). Several overlapping
complementary oligonucleotides are ligated to form one synthetic strand of small
DNA fragment which contains the coding sequence for the first 24 aminoacids of
mature hGH (after removal of the N-terminal signal peptide). The synthetic DNA
and cDNA molecules are ligated to yield a new fragment which contains the
complete coding sequence of hGH. It is ligated into a restriction site just down
stream of the lac promoter / oprater region cloned on a plasmid. The rDNA
plasmids are allowed to transform E.coli. Synthesis of hGH is induced by an
inducer of lac operon (i.e.isopropylthiogalactoside (IPTG)). The hGH is
subsequently purified. hGH is being produced commercially by this method.

About 100,000 molecules
of hormone per cell of E.
coli have been produced
(Newmark, 1979). One of
the major difficulties was
that at the N-end of
polypeptide an extra
methionine, the met-
hGW, was attached.
Methods are developed to
remove methionine from
the met-hGW molecules.
Somatostatin
Somatostatin, a 14
residue polypeptide
hormone is synthesized
in the hypothalamus. It is
the first polypeptide
which was expressed
in E. colias part of the
fusion peptide (Itakura et
at, 1977) which inhibited
the secretion of growth
hormone, glucagon and
insulin. It does not
contain any internal
methionine. Eight single
stranded DNA segments
were synthesized
chemically which were
annealed in an
overlapping manner to
form a double stranded
DNA (the synthetic
gene). It had single



Fig. 5.4. Synthesis of somatostatin, Lac P/O, lactose
promoter/operator, ST, somatostatin; Amp, ampicillin
resistance.

stranded projections at
the each end as the same
are formed by Eco RI.
The synthesized gene
contained 51 base pairs
which were terminated by
two non-sense (stop)
codon and preceded by a
methioine codon as
below:
ATG - (42 base pairs
encoding somatostatin) -
TGATAG.

Two plasmids, p
SOM
I and
p
SOM
11-3, were
constructed. The
synthetic gene was
introduced into E. coli -
galactosidase gene at
different sites.

The chemically synthesized gene was inserted down stream from
the lac promoter in such a way that the gene fusion should have specified a
polypeptide in which the first 7 amino acids of -galactosidase were fused to
somatostatin. But the somatostatin was not detected in transformed bacteria;
possibly it was degraded in E. coli (Glover, 1994). An alternative plasmid,
p
SOM
II-3, was constructed which had the both lac promoter /operater
and lacL regions. The lacZgene encodes peptide of -galactosidase. If the
resulting frame of lacL is maintained after inserting a DNA, a fusion peptide is
produced. The plasmid was cleaved in the lacL segment by restriction enzyme
(Fig. 5.4). The synthetic gene was inserted into the plasmid at Eco RI site near
C-terminus of -galactosidase. The plasmid in the transformed bacterium directs
the synthesis of a fused protein consisting of NH
2
- termined segment of -
galactosidase fragment coupled by methionine to somatostatin. It was stabilized
from proteolytic degradation by -galactosidase moiety (Glover, 1984). This
fused protein was purified and treated with cyanogen bromide (CNBr) which
cleaves only protein at the carboxyl side of the methionine. Thus, the methionine
linker remains attached to -galactosidase fragment, and somatostatin was
released. Now, it has become possible to inhibit the degradation of foreign
protein in E. coli by introduction of protease inhibition (PIN) gene of T4 phage.


endorphin
-endorphin (30 amino acid long neuropeptide with opiate activity) is another
growth hormone which was expressed in genetically engineered E. coli cells.
Shine et al. (1980) integrated DNA sequences of -endorphin, obtained from
mRNA, adjacent to -glactosidase genes on plasmid. The mRNA contained large
precursor of protein that consisted of, besides -endorphin, the hormones -
melanotropin, corticotropin, -lipotropin and -melanotropin. The -endorphin is
cleaved from C-terminus of the precursor peptide. In this way, the transformed
bacteria produced an insoluble fusion protein between -galactosidase and -
endorphin. -endorphin can be removed from the hybrid protein by tripsin which
cleaves only at arginin residue. Before doing so, internal lysines are protected
from trypsinization by citraconylation as below:

NH
3
- -glactosidase - - melanotropin -
endorphin COOH

Citraconylation

Trypsin
- galactosidase + - endorphin