Human Growth Hormone Growth hormone ( - PowerPoint Presentation

Slide1

Human Growth Hormone

Growth hormone (

somatotropin

) size 191 amino acids mol wt. 22125

kd

is secreted by Anterior Pituitary

Growth

hormone stimulates production of insulin-like growth factor 1.

Insulin-like

growth factor 1

is an essential component

of the promotion of growth in children, and

in adults

It

controls metabolism

Infants and

children who

lack sufficient endogenous levels of human growth

hormone

Patients with

chronic renal insufficiency (defective kidneys),

Turner

syndrome

Respond

to treatment with growth

hormone

It stimulates

Tissue

and bone growth,

increases

protein

synthesis

Mineral retention

Decreases

body fat storage

In 1982, human insulin became the first

pharmaceuticals produced by recombinant

DNA technologies

for commercial use.

Since

then, several other

human proteins

with medicinal value have been synthesized in bacteria. Some of the

first human

proteins to be produced in microorganisms were

:

B

lood-clotting

factor

VIII (lacking

in individuals with one type of hemophilia

)

P

lasminogen

activator (a

protein that

disperses blood clots

)

H

uman

growth hormone (a protein deficient in

certain types

of dwarfism).

In 1985,

hGH

became

the second

genetically engineered pharmaceutical

approved

for use in humans

by the

U.S. Food and Drug Administration

.

The

first recombinant growth hormone was called

somatrem

(

Protropin

)

It

had

an amino

acid sequence that was identical to that of human growth

hormone, except

that there was an extra methionine residue at the N-terminal end

of the

peptide

chain

Only growth hormones from humans

or from

closely related primates will function in humans.

Thus

, prior to 1985, the

major source

of growth hormone suitable for treatment of humans was from human

cadavers

hGH

, which is required for normal growth, is a single

polypeptide chain

191 amino acids in length

To obtain expression in

E. coli,

the

hGH

coding sequence

must be placed

under the

control of

E. coli

regulatory elements.

Therefore

, the

hGH

coding sequence

was joined

to the promoter and ribosome-binding sequences of the

E. coli lac

operon

To

accomplish this, a

Hae

III

cleavage site in the nucleotide-pair

triplet specifying

codon 24 of

hGH

was used to fuse a synthetic DNA sequence

encoding amino

acids 1–23 to a partial cDNA sequence encoding amino acids 24–191.

This

unit was then inserted into a plasmid carrying the

lac

regulatory signals and introduced into

E. coli

by transformation

The

hGH

produced in

E. coli

in these first experiments contained methionine

at the

amino terminus (the methionine specified by the ATG initiator codon

).

Native

hGH

has an amino-terminal phenylalanine: a methionine is initially present but

is then

enzymatically removed.

E

. coli

also removes many amino-terminal

methionine residues

posttranslationally

. However, the excision of the

terminal methionine is sequence-dependent

, and

E. coli

cells do not excise the amino-terminal

methionine residue

from

hGH

.

Nevertheless, the

hGH

synthesized in

E. coli

was found to be fully active in humans despite the presence of the extra amino acid.

More recently, a

DNA sequence

encoding a signal peptide (the amino acid sequence required for

transport of

proteins across membranes) has been added to an

h

GH

gene

construct.

With

the signal sequence added,

hGH

is both

secreted and

correctly

processed The

methionine residue is removed with the rest

of the

signal peptide during the transport of the primary translation product

across the

membrane

.

This

product is identical to native

hGH

.

in

2004, the

U.S. Food and Drug Administration (FDA) approved the use of

recombinant human

growth hormone for individuals whose

short stature was

caused by a variety of medical conditions other than human growth

hormone deficiency

.

Native

human growth hormone binds to

both growth

hormone and prolactin receptors that occur on a number of

different cell

types.

To

avoid unwanted side effects during therapy, it is

desirable that

human growth hormone bind only to growth hormone receptors.

Site-specific

mutagenesis of the cloned human growth hormone

cDNA was

used to change some of the amino acid side chains that act as

ligands for

Zn

2+

(i.e., His-18, His-21, and Glu-174), because the ion is required

for the

high-affinity binding of human growth hormone to the

prolactin Receptor

These

modifications yielded human

growth hormone

derivatives that bound to the growth hormone receptor but not

to the

prolactin receptor.

Short

half-life in plasma,

human growth

hormone therapy currently requires subcutaneous injection once

a day

.

This

treatment is both inconvenient and expensive.

The

extracellular domain of the human growth

hormone receptor

was fused to human growth hormone

This

construct has a very strong tendency to

dimerize

as the

growth hormone

moiety from one molecule binds with the receptor portion

of another

molecule.

When

this growth hormone construct was tested in

rats, a

single injection promoted growth for 10 days

Another method that has been devised to prolong the active lifetime

of human

growth hormone includes fusing the coding sequences for

the C-terminal

end of human growth hormone

with the

N-terminal end

of human serum

albumin.

This fusion protein is

called

Albutropin

The

stabilization of the human growth hormone portion of

Albutropin

reflects

the stability of human serum albumin, which has a half-life

in serum

of about 19 days.