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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, let’s 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, β-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 β-galactosidase. When this linked gene is expressed in bacteria, a fusion protein is made between somatostatin and β-galactosidase. The fusion protein is not rapidly degraded. The researchers could then separate somatostatin from β-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 β-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 βgalactosidase 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) 8–320* <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