Recombinant Somatotropin

RECOMBINANT SOMATOTROPIN

Somatotropin, also known as human growth hormone (hGH), consists of 191 amino acids; it is produced in the pituitary gland and regulates growth and development. Regular injections of hGH are given to children with dwarfism caused by the lack of this hormone so that they can reach near normal heights. In this case, unlike with insulin, animal-derived hormones are ineffective and only the human protein works. Because of continuous shortages of pituitaries from human cadavers, the production of recombinant hGH has been imperative. Furthermore, the contamination of children with fatal viruses from the cadavers has been an additional reason for moving away from this source of hormone. As the gene for the hGH is 573 nucleotides long, it was in practice too large to be synthetically produced to generate the recombinant hormone as achieved with insulin and somatostatin. Hence there have been two practical ways of generating recombinant hGH, one of which resulted in the generation of this hormone with an added methionine at the N-terminus. Figure 25.10 shows these two strategies.

Initially the coding region for hGH was isolated from a cDNA library. The DNA fragment coding for the mammalian signal peptide can then be excised by a restriction enzyme that also removes the first 24 codons of the mature protein. A chemically synthesized DNA fragment containing a methionine codon, to enable translation in E. coli, followed by these first 24 codons was therefore ligated to the DNA fragment coding for the remaining amino acids 25–191 of the hGH (Figure 25.10A). The resulting DNA was cloned into an expression vector and transformed into E. coli where the recombinant hGH accumulated in the cytoplasm. The recombinant hormone can be isolated from bacterial cell extracts and, in contrast to the non-recombinant protein, carries a methionine residue at the N-terminus.

Figure 25.10B shows an alternative method consisting of the replacement of the mammalian signal peptide for a secretion signal peptide functional in bacteria. This enables the purification of the recombinant hGH from the periplasm of the bacterial cell, reducing the difficulties associated with the purification of recombinant proteins from the cytoplasm. To achieve this, once the mammalian signal peptide has been removed as above, a synthetic DNA molecule containing the missing 24 codons of the hGH, without an added methionine codon, is ligated to the DNA fragment coding for the 25–191 remaining residues. The resulting DNA molecule is then inserted in an expression vector that codes for a bacterial signal peptide in fusion with the hGH gene. Once transformed into E. coli, the recombinant hGH is produced and the signal sequence will target the protein for secretion into the periplasmic space where it will accumulate. The periplasmic proteases release the signal peptide, leaving hGH without additional amino acids. The protein can then be easily purified from the periplasmic space after release by hypotonic disruption of the outer membrane.