Periplasmic production of correctly processed human growth hormone in Escherichia coli: natural and bacterial signal sequences are interchangeable.

We have studied the synthesis, secretion, and processing of human growth hormone (hGH) in Escherichia coli transformed with plasmids engineered for the expression of hGH as a secreted product. In one plasmid, pPreHGH207-2, the coding sequence of the natural hGH precursor (pre-hGH) is placed under the control of the E. coli trp promoter. In a second plasmid, pAPH-1, a DNA fragment containing the E. coli alkaline phosphatase promoter and signal sequence codons is fused to the mature hGH coding sequence (pho-hGH). Most of the hGH was present in the osmotic shock fluids of E. coli cells containing either plasmid, indicating transport to the periplasmic space. Amino acid sequencing of the N termini of the pre-hGH and pho-hGH gene products revealed that both were processed correctly. Electrophoretic analysis of these polypeptides on reducing and nonreducing sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels indicates that periplasmic hGH is monomeric and contains the same two disulfide bonds as authentic hGH.

Cloning, nucleotide sequence, and expression in Escherichia coli of the exotoxin A structural gene of Pseudomonas aeruginosa.

A 2760-base pair DNA segment of the Pseudomonas aeruginosa strain PA103 chromosome encoding the exotoxin A (ETA) structural gene has been cloned in Escherichia coli and the nucleotide sequence has been determined. Analysis of the 5'- and 3'-flanking regions indicate that ETA is translated from a monocistronic message. Comparison of the deduced NH2-terminal amino acid sequence with that determined by sequence analysis of the secreted protein indicates that ETA is made as a 638 amino acid precursor from which a highly hydrophobic leader peptide of 25 amino acids is removed during the secretion process. Data are presented that indicate a COOH-terminal location of the ADP-ribosylation activity of the molecule. Expression of the ETA coding sequence in E. coli under control of the E. coli trp promoter, but not the ETA promoter, results in the production of enzymatically and immunologically active protein. However, most of this material appears to be neither processed nor secreted. Comparison of the ETA amino acid and nucleotide sequences to those of diphtheria toxin revealed no significant homologies, indicating that these functionally similar toxins evolved from different genes.