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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 t