bioe 110 - hgh (1)
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Recombinant Human Growth Hormone
BioE 110 -Project 1
Due: October 5, 2010
Steven Grillo, Rachel Nordberg, Ilan Beitscher, Peter Sords
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1. Introduction
With the advent of recombinant DNA technology, the controlled
microbial production of an enormous variety of useful polypeptides has
become possible. One such product of the technology that is currently onthe market as well as in use is the recombinant human growth hormone.
Somatropin, or more commonly known as recombinant human growth
hormone (rHGH), is a synthetic growth hormone. Somatropin is a protein
hormone that stimulates growth in the human body along with cell
reproduction and regeneration. Many years ago, inserting HGH into your
body was an ethical and health issue, which discouraged the use by many
people. The health issue was the more significant concern in that the
hormone was attained through the pituitary gland of a cadaver, and hence,
caused infection and disease. Biotechnology became the answer to this
problem. Recombinant DNA technology was used to create the recombinant
human growth hormone. It took until 1985 for Somatropin to replace
pituitary derived HGH. Today, all human growth hormone that is being used
is created via recombinant DNA technology.
The first use of recombinant HGH on humans occurred in 1981 by
Genentech. However, there is an extremely interesting case that developed
in the years of Genetechs initial creation. Allegedly, Genetech stole
research materials that were in the laboratories at the University of
California-San Francisco. They were blamed of sneaking into their
laboratories at midnight on New Years Eve and simply stealing materials
from the Universitys scientists. There was an extremely long trial that took
place and ended in Genetech having to pay the University $150 million and
donate $50 million towards a new research building. This case led to larger
arguments about genes and gene sequences and their patentability
(Rimmer).
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Somatropin, identical to the growth hormones in the human body, is a
22,000 Dalton and 191 amino acid sequence peptide produced during
fermentation in E.coli. Initially a 192 amino acid sequence is formed, but
the methionine is removed to create an exact match to the pituitary derived
human growth hormone. The growth hormone works by a process of
secretion by the somatotrophs of the anterior pituitary gland. It then
enables growth within the body due to interaction with various tissues.
Synthetic HGH is used to treat individuals who suffer from growth
hormone deficiency. HGH has many functions including tissue repair, muscle
growth, bone strength, brain function, and metabolism. An individual with a
deficiency of growth hormone can be abnormally short in stature and
maintain a slow growth rate and is therefore diagnosed with pituitary
dwarfism. There are many cases in society in which a doctor would
recommend that a child
take human growth
hormone so that the
individual can have the
ability to be at a more
typically normal height.
HGH is produced by
means of recombinant
DNA technology and is
used for treatment of
pituitary dwarfism in the
field of pediatrics.
However, synthetic human growth hormone is misused greatly by athletes
and others around the globe. HGH is not supposed to act as enhancement
therapy or as a booster for additional growth for an individual who is
already considered or projected to be normal height. Unfortunately, it is not
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the case that this is only used for treatment. This is reflected by the
numerous doping or steroid problems within professional sports. Athletes
use HGH to enhance their individual performance in their respective sports
to gain a competitive advantage.
HGH is currently on the market and is a drug that should be prescribed
by a medical doctor. As of 2005, recombinant growth hormones available
in the United States included Nutropin (Genentech), Humatrope (Lilly),
Genotropin (Pfizer), Norditropin (Novo), and Saizen (Merck Serono). This is
a drug that has an affect on humans each and everyday. It can be a minute
reason such as a favorite baseball player caught doing steroids or a more
serious scenario if you are a person that may be suffering from human
growth hormone deficiency and need to consider taking HGH. Somatropin
treats a variety of conditions of growth failure and growth retardation. A
major group of people in which rHGH can help are those who suffer from
growth hormone deficiency, Turners syndrome, and short stature according
to skeletal age. Recombinant DNA technology opens the way to the large-
scale commercial production of human growth hormone, and the
recombinant HGH appears to have equivalent biological efficacies and
pharmacokinetic properties. Recombinant technology has led to advances
in protein production and classification, and therefore has increased the
therapeutic applications of proteins and will hopefully continue to have an
impact in the future.
2. Biotechnology
2.1 Characterization of Human Growth Hormone
Human growth hormone (hGH) is secreted in the human pituitary, a pea-
sized gland located at the base of the brain. The molecular weight of hGH is
about 22,000 (22K form) and it consists of 191 amino acids (or residues)
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with two loops formed by disulfide bridges (see
Figure 6). The molecule is predominantly -
helical in secondary structure.
Involved in metabolism, growth, and lactation,
GH can have both anabolic and catabolic
effects: while it increases lean mass by
increasing muscle and bones, it reduces fat.
Some effects are mediated directly, others are
mediated indirectly through the action of
somatomedins hGH-dependent growth
factorssuch as insulin growth factor 1 (IGF-1). Figure 1 illustrates hGH
(top left) and IGF-1 (bottom) proteins,
along with prolactin (top right), another
pituitary hormone closely related to
hGh. Because of its pervasive role in the
growth of both soft and skeletal tissue,
human growth hormone is of
considerable medical importance, and
its cloning will provide boundless
benefits.
2.2 The construction and expression
of a cloning vehicle for human
growth hormone
Since hGH is a non-glycosylated protein, prokaryotic expression systems
such as Escherichia coli have been preferred in the production of
recombinant hGH. In 1979, scientists at Genetech produced human growth
hormone by inserting DNA coding for human growth into a plasmid that was
Figure 1
Figure 2
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implanted in E. colibacteria (Goeddel and Heyneker). This was first done the
following way:
I. Cloning the Hae III fragment of the mRNA transcript (Figure 2)Polyadenylated mRNA for human growth hormone was prepared from
pituitary growth hormone tissue. A double strand (ds) cDNA was prepared
from this RNA. The restriction pattern of hGH is such that Hae III restriction
sites are present in th 3 noncoding region and in the sequence coding for
amino acids 23 and 24 of hGH. Treatment of ds hGH cDNA with Hae III gives
a DNA fragment of 551 base pairs (bp) coding for amino acids 24-191 of
hGH. pBR322 was chosen as the cloning vehicle for the cDNA.
Plasmid pBR322, shown in
Figure 3, is a widely used
plasmid and has been
completely sequenced, with
a known size of 4363bp.
The most useful aspect of
the DNA sequence is that it
characterizes pBR322 in
terms of its restriction
sites, such that the exact
length of every fragment
can be calculated. There
are 40 enzymes with
unique cleavage sites on the pBR322 genome. pBR322 is a convenient
cloning vehicle for hGH not only because it is a multicopy replicating
plasmid, but also because it exhibits both ampicillin and teracycline
resistance owing to its inclusion of the corresponding genes (ApR and
Figure 3
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TcR, respectively) and
contains recognition sites for
the restriction enzymes Pst I,
EcoRI and Hind III.
A GC tailing mehod was then
employed to combine products
od Pst I cleavage of pBR322
and of Hae II digestion of the
mRNA transcript, inserting the
cDNA fragment into the Pst I
site of pBR322 in such a
manner as to restore the Hae
III restriction sites on the
cDNA while also restoring the
Pst I restriction sites at each
end of the insert. After annealing of the dC-tailed ds cDNA with the dG-tailed
vector DNA, the mixture was first amplified using PCR, and then used to
transform E. Coli x1776. The resulting plasmid, given the name pHGH31
cloned in x1776 was analyzed using DNA sequence anylisis and was
confirmed to contain the codons for amino acids 24-191 of hGH.
II. Construction and Cloning of the Synthetic Gene Fragments (Figure 4)III. Construction of Plasmid for the Bacterial Expression of hGH (Firgure
5)
With the synthetic fragment in pHGH3 and the mRNA transcript in pHGH31,
a replicatable plasmid containing both fragments was constructed using the
expression plasmid pGH6, shown in Figure 5. The expression plasmid pGH6
containing tandem lac UV5 promoters, was treated successively with Hind
Figure 4
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III, nuclease S1, and Eco RI and purified by gel electrophoresis. The
resulting vector was ligated to the 591 bp hGH DNA.
The ligation mixture was then to transform E. Coli x1776. Colonies were
selected for growth on tetracyclin. It is noteworthy that insertion of the
hybrid hGH gene into pGH6 destroys the promoter for the tetracyclin
resistance gene, but that the tandem lac promoter permits read-through of
the structural gene for tet resistance, retaining this selection characteristic.
Transformants are then obtained and filter hybridization distiguishes colonies
Figure 5
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containing hGH sequences. The DNA sequences of one clone, pHGH107, was
determined.
2.3 Structure of hGH
When engineering a novel protein
for large-scale production in
vitro, structure of the protein
itself is of critical importance. The
factor underlying the proteins
conformation is its amino acid sequence. Figure 6 displays the amino acid
sequence of hGH that any successful recombinant DNA should accurately
translate. Any significant change in amino acid sequence could render the
hGH protein less functional or even completely inactive. For example,
changing a single glycine at position 120 to arginine will inactivate the hGH
by not allowing it to bind to its receptor.
As mentioned before, the experimentally
determined protein structure of hGH
predominantly consists of -helices. More
specifically, hGH contains four helices 21-30
residues long that are arranged in a left-
handed bundle. The topology in unusual inthat the first two helices (beginning at the N-
terminus) are parallel to each other and
antiparellel to the last to (Wells and
Abraham). To achieve this arrangement, long crossover connections link the
Figure 6
Figure 7
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two sets of parallel helices, and a sort segment connects helix 2 to helix 3.
Dilsulfides connect C53 in the first crossover connection to C164 in helix 4,
and C181 in helix 4 to C189 near the C terminus.The core of hGH is almost
exclusively made up of hydrophobic side chains. Figure 7 shows the hGHs
tertiary structure found on Protein DataBank.
2.4 Hormone ligand-receptor complex
Growth hormone, shown in red in Figure 8,
performs its multiple regulatory functions by
binding to growth hormone receptors (shown in
blue and green) on its target organs and cells.
Growth hormone receptors are single-pass
transmembrane receptors, with a three-domain
architecture: an extracellular domain that binds
to the activated ligand, a helical transmembrane
segment, and a domain within the cytoplasm.
Interestingly, growth hormone must bind to two
receptor molecules simultaneously to mediate its funtion. This discovery thatactivation of the GH receptor requires dimerization can be used to produce
receptor-specific analogues and to design receptor agonists and antagonists.
(ex. Antagonist useful for treatment of acromegaly, a disease caused by the
overproduction of hGH secreted from the pituitary.
3. Bioprocessing
3.1 Production of Plasmid
The host cell used for the transformed production of hGH will be E. coli
BL 21. The plasmid pBR322 will be used for the insertion of the hGH gene
(figure 3). The hGH gene will be inserted with the use of the restriction
enzymes EcoRI and HindIII. The restriction enzymes will digest the plasmid
Figure 8
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leaving sticky ends that will then adhere to the hGH gene. The insertion of
the hGH gene would destroy the promoter for the gene that causes the E.
coli to be resistant to tetracycline. Thus, using a tetracycline plate will allow
for separation between the cells with the hGH gene and those without. The
colonies that contain the desired hGH positive vector will be selected for
fermentation.
3.2 Fermentaion of E. Coli cells
In order to create enough E. coli for cultivation of the hGH protein, a
batch reactor growth scheme must be implemented. The fermentation in this
process will take place at a pH of 7 and a temperature of 37C because that
is the ideal condition for E. coli to grow. The pH will be maintained by the
addition of ammonium hydroxide and phosphoric acid, as needed. The feed
into the bioreactor will include glucose as a carbon source, anti-foaming
agents, water, yeast as a nitrogen source, and trace amounts of B-D-
thiogalactopyranoside (IPTG) to induce expression. Furthermore, a mixture
of nutrients, trace metals, and inorganic salts will be added to the mixture in
order to optimize E. coli growth. A mixture such as MaxyBroth manufactured
by BioProgen Co. may be used (Shang). These reagents will all be added to
a stirred tank reactor so that everything will be mixed together evenly.
There will be high purity oxygen bubbled through the solution at
concentrations higher than 30% in order to optimize the E. coli growth
(Shang). The carbon source for growing the E. coli will be glucose, which will
be added to the reactor at a rate slow enough to not exceed the oxygen
supply and consequently, avoid the formation of acetic acid. The outflow of
the reactor will be an E. coli rich solution containing rhGH ready to be
processed.
3.3 Separation of Inclusion Bodies
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After fermentation, the cells must be separated from the batch
fermentation medium. There will be inclusion bodies within the E. coli
because the hGH hormone is very large. In order to isolate the inclusion
bodies a three-step process will be used. First, the E. coli cells will be
isolated from the rest of the mixture by centrifugation for 5 minutes at 5000
rmp. This is a slow speed centrifugation so that the cells will not be
damaged. The E. coli cells are large and hence, they will form a pellet at the
end of the centrifugation. Once the pellet is formed the media will be
decanted off. The cells were then re-suspended in a lysis buffer so that that
there is a clean solution for the cells to release their inclusion bodies. The
inclusion bodies will contain the recombinant hGH. The cells will then be
shocked by rapidly cooling them to 0C. This is done so that the cells will
lyse and inclusion bodies will be released into the solution. The lysis will be
followed by a second centrifugation for 5 minutes at 10,000 rpm in order to
isolate the inclusion bodies for the cell debris. This is a faster centrifugation
because the inclusion bodies are smaller than the cells and will not break as
easily as the cells. Again, the inclusion bodies and other large particles in
solution will form a pellet at the bottom of the centrifugation tube and theliquid media will need to be decanted.
3.4 Processing of rhGH protein
The inclusion bodies containing rhGH will be suspended in a tris-HCl
buffer solution. The inclusion bodies will go through chemical conversions in
order to become the desired protein conformation of rhGH. In order to do
this, a cleavage reaction will be induced using urea and guanidine
hydrochloride because rhGH is a fusion protein. As a fusion protein, multiple
genes were artificially combined to code for the protein. The two parts of the
protein must be separated and thus will be cleaved apart during the
reaction. Anion exchange chromatography will be used to separate the
proteins knowing that rhGH is negatively charged. Once the proteins are
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separated, sulfitolysis will be used to reform the disulfide bonds. This
process will be done at a pH of 7 at room temperature because rhGH is
highly sensitive to heat. Heat will cause the protein to denature again so the
renaturing of the protein must not be in a heated system. Diafiltration will
then be used to remove small ions and molecules left over from the chemical
conversion steps. The diafiltration will get rid of some of the excess ions and
molecules leaving the protein of interest and larger molecules. The rhGH will
then be extracted from the diafiltration device and be ready to start
chromatography.
3.6 Chromatography Methods
Once the protein is refolded, a series of chromatography methods will
be used to separate the protein from the impurities. First, a size exclusion
gel filtration chromatography will be used to remove small fragments and
salts in the solution and to let the protein pass. The ions and small debris
will be trapped in the matrix while larger molecules like the target protein
rhGH will be able to cross the gel. Once the solution passes through the
column, the lipids, glucose, and trace amounts of ions will still be in the
solution with the rhGH. Hydrophobic interaction chromatography will help
remove salts and cause hydrophobic non-polar fat molecules to aggregate.
To completely
separate hydrophobic
and hydrophilic
proteins, a reverse
phase chromatography
is performed. The
hydrophilic outer
portion of rhGH will
not bind to theFigure 9
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stationary hydrophobic phase and therefore remove other hydrophobic
proteins from the mixture. In order to separate the protein from the rest of
the solution ion exchange chromatography will be used. The rhGH protein is
negatively charged. Therefore, an anion exchange using a positively
charged column will capture the rhGH, but allow other impurities to flow
through. The hydrophilic functional groups on the outside of rhGH will
interact with the charged beads in the column. The nonpolar lipids and
sugars will go straight through the column while the hGH protein stays in the
column as the stationary phase. Ultrafiltration, the final filtration process,
will suspend the large molecule of rhGH while allowing any remaining salt,
water, and minerals left in solution to pass through the membrane. Figure 9
shows what a protein gel might look like after various stages of
chromatography with A being prior to chromatography and C being after
many stages of chromatography. Once the rhGH is pure, it is ready to be
recrystallized for storage.
3.7 Recrystallization of rhGH protein
After filtration is complete a recrystallization process must be carried
out to make the biosynthetic drug a solid. Although at this time insulin is the
only human therapeutic protein that is crystallized for administration, there
are many advantages to the recrystallization of the protein such as extended
shelf life and increased potency (Crisman). The recrystallization, like the
renaturing steps earlier, will need to be preformed at room temperature so
that the protein does not denature again. A nucleation agent will be added to
the rhGH protein and the protein will become a solid, which will be able to
last on the shelf for months until it is administered for clinical use.
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