Robert J.

Brooker - Genetica

Esperimento di genetica 19.1

Somatostatin Was the First Human
Peptide Hormone Produced
by Recombinant Bacteria
During the 1970s, geneticists became aware of the great potential of recombinant DNA technology to produce therapeutic agents
to treat certain human diseases. Healthy individuals possess many
different genes that encode short peptide and longer polypeptide
hormones. Diseases can result when an individual is unable to produce these hormones.
In 1976, Robert Swanson and Herbert Boyer formed Genentech
Inc. The aspiration of this company was to engineer bacteria to synthesize useful products, particularly peptide and polypeptide hormones. Their first contract was with researchers Keiichi Itakura and
Arthur Riggs. Their intent was to engineer a bacterial strain that
would produce somatostatin, a human hormone that inhibits the
secretion of a number of other hormones, including growth hormone, insulin, and glucagon. Somatostatin was not chosen for its
commercial potential. Instead, it was chosen because the researchers thought it would be technically less difficult than other hormones. Somatostatin is very small (only 14 amino acids long),
which requires a short coding sequence, and it can be detected easily.
Before discussing the details of this experiment, lets consider
the researchers approach to constructing the somatostatin gene. To
express somatostatin in bacteria, the coding sequence for somatostatin must be inserted next to a bacterial promoter that is contained within a plasmid. Rather than obtaining the gene from the
human genome that encodes this 14-amino acid hormone, the researchers took a different approach. As shown below, they chemically synthesized short (single-stranded) oligonucleotides that
would hydrogen bond with each other to form the coding sequence
for the gene shown in Figure EG19.1.1.
Eight separate oligonucleotides (labeled A through H) were synthesized chemically. Due to base complementarity within their sequences, the oligonucleotides hydrogen bonded to each other forming a longer double-stranded DNA fragment with two important
characteristics. First, its single-stranded ends (i.e., overhangs) al-

lowed it to be inserted into EcoRI and BamHI restriction sites

within plasmid DNA. Second, the middle of this DNA fragment
encodes the amino acid sequence of the somatostatin peptide hormone. (Today, oligonucleotide synthesis methods have greatly improved, making it unnecessary to synthesize several, short oligonucleotides. Instead, researchers now could make an oligonucleotide
that would span the entire length of the somatostatin coding sequence.)
The coding sequence was constructed so that an extra methionine would be located at the amino terminal end of somatostatin.
This methionine provided a link between somatostatin and a bacterial protein, -galactosidase. As discussed in Chapter 14, this enzyme is encoded by the lacZ gene. Why was this link necessary?
During the course of their experiments, the researchers learned that
somatostatin made in bacteria is rapidly degraded by cellular proteases. To prevent this from happening, they linked the somatostatin
sequence to the lacZ gene encoding -galactosidase. When this
linked gene is expressed in bacteria, a fusion protein is made between somatostatin and -galactosidase. The fusion protein is not
rapidly degraded. The researchers could then separate somatostatin
from -galactosidase by treatment with cyanogen bromide (CNBr),
which cleaves polypeptides at the carboxyl terminal side of methionine. Because no methionines are found within the somatostatin
sequence itself, this treatment does not degrade somatostatin.

The steps in their protocol are shown in Figure EG19.1.2. As described here, the researchers made a synthetic somatostatin gene
that was flanked by unique restriction sites and had a methionine

Figure EG19.1.1

2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia

Robert J. Brooker - Genetica

codon at the beginning of the somatostatin-coding sequence. This

gene was then inserted into a plasmid at the end of the lacZ gene.
As a control, they also inserted the somatostatin gene in the wrong
orientation (shown on the right of step 3). The plasmid with the
wrong orientation should not make any somatostatin. The plasmids
were then introduced into E. coli cells. The plasmid contained the
lac promoter (from the lac operon), which was induced with IPTG
(a nonmetabolizable lactose analogue). In the cells harboring the
somatostatin gene in the correct orientation, this would induce the
synthesis of a fusion protein containing -galactosidase and somatostatin. The bacterial cells were then collected by centrifugation

and exposed to formic acid and cyanogen bromide (CNBr). As

mentioned, the CNBr cleaves polypeptides next to methionine residues. Therefore, this treatment would break the link between galactosidase and somatostatin. The amount of somatostatin was
then determined by a radioimmunoassay (RIA). (See the Appendix
for a description of radioimmunoassay.)

THE GOAL
The researchers wanted to produce human somatostatin in a recombinant bacterium.

Starting material: A normal E. coli strain that was unable to synthesize somatostatin, and bacterial plasmids that carry the ampR gene

FIGURE EG19.1.2 The production of human somatostatin in E. coli.

2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia

Robert J. Brooker - Genetica

2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia

Robert J. Brooker - Genetica

THE DATA
Plasmid Strain
Correct orientation
Incorrect orientation

INTERPRETING THE DATA

Amount of Somatostatin (detected by RIA)
(picograms of somatostatin/
milligram of bacterial proteins)
8320*
<0.4

*The amount of somatostatin was determined in several independent experiments.

As shown in the data table, recombinant bacteria carrying the somatostatin gene in the correct orientation produced this hormone. The
amount of somatostatin varied from 8 to 320 picograms per milligram of bacterial proteins. This variability could be attributed to
several factors, including protein degradation, incomplete cyanogen
bromide cleavage, and unknown genetic changes in the plasmids
during bacterial cell growth. In spite of this variability, the exciting
result was the production of a human hormone in recombinant bacteria. By comparison, the plasmid with the incorrect orientation did
not produce a significant amount of the hormone. This study was
the first demonstration that recombinant bacteria could make products encoded by human genes. At the time, this was a major breakthrough that catalyzed the growth of the biotechnology industry!

2010 The McGraw-Hill Companies, S.r.l. - Publishing Group Italia