3.13 Let's Clone!

We now may use the various procedures that we have learned to examine making a plasmid vector, transforming cells and producing human polypeptides in E. coli.

Somatostatin (Itakura, et al., Milestones readings, p. 84)

Somatostatin is a very small peptide hormone and provides a nice example of the major principles to be considered when cloning a gene (see figure 25). The polypeptide amino acid sequence is known, is only 14 amino acids long, and contains one internal disulfide bond. The homone is produced in the pancreas and other tissues and inhibits the secretion of insulin and glucagon. It also reverses glucagon effects in the liver.

FIGURE 25. Cloning somatostatin

The DNA gene was synthesized chemically after deciding likely codons for each amino acid. Notice that deoxynucleotides A through H were prepared so that they would anneal with one another in an overlapping sequence that could be sealed by ligase. Also notice the EcoRI site synthesized at one end of the molecule and a BamHI at the other. When we open pBR322 using these two restriction endonucleases, a segment of the plasmid is cleaved out and discarded and our new gene will enter the opened plasmid in only one orientation. Notice also that the lac promoter and control region and part of the b-galactosidase gene have been inserted into the plasmid. This means that the protein will not be produced until an inducer has been added to the medium and that the protein product will consist of part of the b-galactosidase protein linked to the polypeptide that we want.

Figure 26. Strategy for the chemical synthesis of the gene for stomatostatin

Another trick that was used was to insert the codon for methionine as the first amino acid in front of the code for somatostatin. Somatostatin itself does not contain methionine. Cyanogen bromide may be used to chemically break the polypeptide bond at all methionine residues, thus liberating the polypeptide from the beta-galactosidase-somatostatin protein product. Two stop signals were inserted into the gene at the end of the somatostatin gene sequence to ensure that protein synthesis would not continue beyond this point. Recombinant E. coli colonies were selected as usual using antibiotic resistance markers, and clones yielding the highest amounts of somatosatin were further screened with a radioimmune assay. The protein product that contained somatostatin was found to reach over 3% of the total cellular protein in some of the clones. The peptide hormone was successfully purified from the transformed cell.

 

Insulin (Crea, et al., 1978. Proc. Nat. Acad Sci., 75:5765)

Almost the same procedure as just described was used for the first cloning of insulin. In this case, synthetic DNA sequences were prepared for both the A (77 base pairs) and B (104 base pairs) polypeptide chains, and these were inserted separately into plasmids containing lac sequences. The individual vectors could be used to transform separate cultures of E. coli. After replica plating and selection, clones were found that produced high yields of either the A- or the B-chain. The insulin polypeptide chains also do not contain methionine, so that cyanogen bromide could be used to cleave out the mature polypeptides from proteins produced in transformed cells. The A- and B-chains were purified away from E. coli cellular material and beta-galactosidase fragments, and conditions were worked out so that when the two purified chains were incubated together under reducing conditions, mature insulin was formed with high yield and with the disulfide bonds in the correct position.

Figure 27. Cloning insulin