Salvatore Toma

A new human growth hormone production process using a recombinant Bacillus subtilis strain

We constructed a series of hybrid plasmids which directed the synthesis of different human growth hormone (hGH) precursor sequences in Bacillus subtilis. In addition to the 191 amino acids of the hormone, the precursors had in common an amino-terminal extension characterized by the presence of a methionine at position 1 and of the tetrapeptide Ile-Glu-Gly-Arg preceding the first residue (Phe) of hGH. The sequence between the methionine and the tetrapeptide was specific for each precursor and, because of the presence of charged residues, conferred particular properties to the molecules. Long homopolymeric tail-containing precursors such as MRRRRRRIILM-IEGR appeared insoluble whereas shorter sequences of the type MRR-IEGR and MEELM-IEGR augmented the solubility of the precursors with respect to Met-hGH. The soluble precursors could be easily purified from the bulk proteins taking advantage of the charged residues present on the N-terminal tail. After purification, the natural hGH was obtained by treating the precursors with the protease Factor Xa which cleaves after the arginine residue of the tetrapeptide IEGR. A protocol for the production and purification of authentic hGH from a strain expressing one of these soluble precursors is reported.

New plasmid expression vectors for Bacillus subtilis

The construction of new cloning vectors for Bacillus subtilis is described. They are derived from the in vitro joining of parts of pE194 and pUB110 DNAs. Their common feature is to present a cloning site immediately after the promoter and ribosome binding site of the erythromycin resistance gene, allowing the insertion and expression of either sticky or blunt ended DNA fragments coding for any heterologous gene. The cloning and expression of Escherichia coli beta-lactamase and EcoRI methylase are given as examples. The enzymes are efficiently synthesized by B. subtilis cells.