TRANSLOCATION AND PROCESSING OF VARIOUS HUMAN PARATHYROID-HORMONE PEPTIDES IN ESCHERICHIA-COLI ARE DIFFERENTIALLY AFFECTED BY PROTEIN-A-SIGNAL-SEQUENCE MUTATIONS

Two staphylococcal protein-A signal sequences were constructed and tes ted for function in Escherichia coli, after being linked to human para thyroid hormone (hPTH) cDNAS representing the intact form (1-84 amino acids) and two N-terminal (1-37 and 1-7 amino acids) peptides. One sig nal sequence was identical to the wild type, and the other signal cont ained a deletion of 12 bp at the 3' end. The truncated hPTH cDNAs were fused at their 3' ends to IgG-binding domains (ZZ) derived from prote in A in order to facilitate purification and characterization. The exp ression plas mid pSPTH, containing the wild-type signal sequence, secr eted efficiently the intact recombinant hPTH (1-84) into the medium. P lasmids containing the truncated hPTH genes after the wild-type signal , gave rise to hPTH-ZZ hybrid proteins which were correctly processed at the N-terminal, but the major fractions appeared in the periplasmic compartment. In contrast, the plasmid pS'PTH which harboured the 4-am ino-acid signal deletion did not promote a uniform secretion of intact hPTH (1-84) to the medium, but released a non-processed form both int o the periplasmic compartment and to the medium. The related plasmids pS'PTH37ZZ and pS'PTH7ZZ with the mutated signal sequence gave rise to small or trace amounts of unprocessed forms of fusion proteins in the medium and periplasm, thus the secretion competence was markedly redu ced. Thus, for correct N-terminal processing, we conclude that the ami no acid sequence in the signal adjacent to the expressed protein, is a key determinant. However, release into the medium or periplasmic spac e appeared to be dependent also on protein folding, irrespective of si gnal-sequence cleavage. Furthermore, we observed that the peptides wit h the wild-type signal sequence and correct N-terminal processing, wer e the only forms that showed internal cleavage of hPTH. Uncleaved sign als may contribute to folding characteristics of the ensuing protein a nd e.g., prevent internal proteolysis.