STAGES IN SOMATOSTATIN SYNTHESIS:

1. The promoter sequence from the lac operon was inserted into pBR322 plasmid by cutting,

insertion and ligation using ligase enzyme.

2. Two superfluous EcoRI sites were then removed (to prevent circle falling apart when this

restriction enzyme was used in the next stage). This produced a smaller plasmid called pBH 20.

3. The chemically synthesized somatostatin gene was then inserted into the plasmid by removing

a small fragment using two restriction enzymes (EcoRI and BamHI) followed by ligation. The

plasmid now looked like this:

4. E. coli cells were then transformed and transfected with the plasmid. But on growth, no

somatostatin production was detected! The reasons for this failure were not immediately

obvious.

In E. coli small peptides are degraded. Somatostatin was actually being synthesized by the host

cells but they contained enzymes which were then digesting the product .

5. Plasmid was then reconstructed to replace the lac operon with a larger fragment containing the

promoter and most of the lac z (-galactosidase) gene. This made the product larger and,

therefore, not prone to degradation by host cell enzymes. The reading frame now needed

correction.

6. On transformation and transfection of E. coli, a "fused" product was produced which consisted

of part of the -galactosidase product attached to the somatostatin peptide.

7. The fused product was then separated into its two component parts using cyanogen bromide

which cuts the amino acid sequence at a methionine between the somatostatin and -

galactosidase chains.