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To obtain an expression vector that will optimize secretion of proteins with disulfide bridges in Escherichia coli, we fused the phoA gene, encoding the bacterial alkaline phosphatase (PhoA), to the sequence encoding the pectate lyase B signal sequence (PelBSS). We used an extensively degenerate pelBSS with silent mutations to study their effects on the production level and activity of PhoA. 11 representative clones differed by a factor of five between the lowest and the highest level of activity, and by a factor greater than seven for the production levels. The efficiency of translocation seems to be the result of an equilibrium between production and secretion levels that favours the secretion of active PhoA according to the competence of the fusion protein being translocated. Free energy calculations and the predicted mRNA secondary structures of the translation initiation regions showed that the high stability of the secondary structure decreased production and secretion levels of PhoA and vice versa. A stem-loop encompassing the degenerate positions downstream from the AUG start codon appears to be responsible for the differences in the production levels.

We have studied the synthesis, processing and export of human growth-hormone-releasing factor (hGRF) in Escherichia coli transformed with a plasmid constructed for the expression of hGRF as a hybrid protein. A DNA fragment containing the entire sequence of phosphate-binding protein gene (phoS) is fused to a modified hGRF-coding sequence (phoS-mhGRF). The hybrid protein, PhoS-mhGRF, was recovered in the supernatant fluid after spheroplasting treatment indicating correct export to the periplasmic space. Pulse-chase experiments demonstrated that the hybrid protein was similarly processed as the PhoS precursor.

Ovalbumin is secreted by the tubular gland cells without cleavage of a signal sequence at the N-terminus. In Escherichia coli strains which produce a chicken ovalbumin-like protein (OLP) from a plasmid-cloned gene, the OLP is synthesized on membrane-bound polysomes and secreted without cleavage into the periplasmic space. In contrast, a deleted protein, which lacks 126 amino acids in the N-terminal half, is not secreted and is synthesized from free polysomes. Our results are compatible with the presence, in the N-terminal half of the molecule, of a signal sequence necessary for the transport across the membrane.

DNA constructs coding for human growth hormone (hGH)-releasing factor (hGRF) preceded by the specific recognition sequence for the activated blood coagulation factor X (FXa), fused in frame to the N-terminal 172-amino acid residues of colicin A, have been expressed in Escherichia coli. The construct was placed under the control of the inducible caa promoter in an operon containing a downstream gene coding for the cell lysis protein, Cal. Induction resulted in excretion of only the processed colicin A fragment. Replacement of Cal by the terminator from phage fd resulted in high expression of the hybrid protein, which was recovered as cytoplasmic aggregates. Enzymatic cleavage of the purified and renatured hybrid protein using FXa allowed the recovery of authentic hGRF.