hGH is a protein of 191 amino acids produced in the anterior lobe of the hypophysis throughout the life of an individual, and in greater quantities during the preadult period.
The growth hormone is synthesised in the form of a precursor and, once processed is secreted from the cell.
hGH has for some time been used for curing some forms of dwarfism, which are due to lack of the hormone, and can also be used in the treatment of obesity and for healing burns and wounds.
Until a few years ago, the only source of the hormone was the hypophyses of corpses from which it was extracted at low yields by a complex and expensive method.
Methods have recently been developed for the preparation of hGH by means of fermentation, with the use of host organisms transformed by recombinant DNA techniques.
In particular, GB patent no. 2055982 and EP patent no. 20147 described and claim a method for the preparation of hGH with the use of an engineered strain of Escherichia coli (E. coli) containing a plasmid which includes the structural sequence of the DNA which codes for the mature protein of 191 amino acids. The sequence is located downstream of a promoter and a ribosome recognition site (RBS) which are necessary for the transcription and translation processes, and juxtaposed to the ATG triplet which codes for methionine.
The presence of the methionine is necessary since it represents the starting signal for the translation of the whole protein.
Known methods, therefore produce a product which is constituted by the amino acid sequence of natural hGH with a methionine (Met) on its amino-terminal.
Although the presence of this amino acid does not seem to affect the activity of the hormone (Olson K. C. et al. (1981), Nature 293, 408), immunological data show that in a fairly high percentage of patients treated with the Met:hGH the appearance of antibodies against the hormone, which prevent extended therapeutic treatment, can be shown.
There is consequently a need to provide a protein identical to the natural one.
Various methods have been proposed in the art for the removal in vivo and in vitro of methionine from the recombinant proteins, and in particular from hGH. On of the in vivo methods is based essentially on the use of signal sequences responsible for the activation of the transport of proteins through the cell membrane of Escherichia coli (E. coli) or Bacillus subtilis (B. subtilis).
In general this method comprises the construction of hybrid plasmids containing the nucleotide sequence which codes for mature hGH, fused at its 5' terminal to a sequence which codes for a signal sequence (a leader sequence) and the transformation of host cells by the hybrid plasmids. Suitably grown, the cells produce a fused protein, that is a protein constituted by hGH and the signal sequence. The sequence is then removed at the membrane level by a specific endopeptidase and the mature protein released into the periplasmic space or into the outside environment. In particular, the leader sequences of the protein OmpA (Hsiung H. M. et al. (1986) Biotechnology 4, 991) and the endotoxin of E. coli were used for the preparation of mature hGH from transformed E. coli cells and the signal sequence of the neutral protease of B. amyloliquefaciens was used for the preparation of hGH from B. subtilis cells.
However, these known methods have disadvantages resulting on the one hand from the use of E. coli, which is a pathogenic organism in man, and on the other hand from the production of an hGH containing from 1 to 4 residual amino acids at its amino-terminus due to the partial processing of the protein expressed by B. subtilis.
A second method for removal of the methionine in vitro consists of modification of the gene which codes for a protein so that its synthesis product is constituted by the natural protein and by a longer or shorter peptide sequence which can be removed by treatment with enzymes. In general, this method suffers from the fact that if residual amino acids, which are subject to attack by the enzymes used for the hydrolysis are present in the protein concerned, a greater or lesser percentage of the protein itself is degraded, consequently lowering the production yield.
It is therefore fundamental to provide a specific treatment which enables the fused protein to be hydrolysed exclusively at the point of connection between the product of interest and the amino-terminal peptide.
It is known that Factor Xa, a blood serine protease involved in the complex process of clotting, recognises the Glycine-Arginine (Gly-Arg) sequence, and in particular has a very great affinity for the isoleucine-glutamic acid-glycine-arginine (Ile-Glu-Gly-Arg) tetrapeptide.
Recently, Nagai and collaborators (Nagai, K. et al (1985), Proc. Natl. Acad. Sci. USA 82,7252 have shown that fused products, in which the amino-terminal portion had been positioned to protect the protein of interest from the action of microbial proteases, were processed correctly and specifically when the Ile-Glu-Gly-Arg sequence was positioned at the connecting point.
Whilst enabling the correct processing of the fused proteins and therefore the production of hGH in the natural form, these known methods however have disadvantages resulting from the difficulty of purification of the product obtained.
In fact, the precursor of hGH is difficult to separate both from the pool of proteins produced by the recombinant cells and from the hormone obtained after enzymatic hydrolysis.