No abstract

The nucleotide sequence of the gene (pac) coding for penicillin G acylase from E. coli ATCC 11105 was determined and correlated with the primary structure of the two constituent subunits of this enzyme. The pac gene open reading frame consists of four structural domains: Nucleotide positions 1-78 coding for a signal peptide, positions 79-705 coding for the alpha subunit, positions 706-867 coding for a spacer peptide, and positions 868-2538 coding for the beta subunit. Plasmids were constructed which direct the synthesis of a pac gene product lacking the signal peptide, and the synthesis of the alpha subunit or the beta subunit. The following results were obtained: The two dissimilar subunits are processing products of a single precursor polypeptide; the spacer peptide is removed during processing; the precursor polypeptide lacking the signal sequence is accumulated in the cytoplasm; it is not processed proteolytically in the cytoplasm and it does not display enzyme activity. Processing, therefore, requires translocation through the cytoplasmic membrane; processing follows a distinct sequential pathway in vitro.

No abstract

Using standard growth conditions (LB medium, 37 degrees C, induction with 5 mM IPTG) yeast alpha-glucosidase PI expressed under the control of the regulated tac-hybrid promoter results in the synthesis of insoluble aggregated alpha-glucosidase granules in Escherichia coli. Under these conditions active soluble alpha-glucosidase amounts to less than 1% of the heterologously produced protein. However, the amount of soluble active alpha-glucosidase was dramatically increased when the strong tac-hybrid promoter was to a limited extent induced. This was achieved at concentrations of 0.01 mM IPTG or of 1% lactose or lower in a lactose-permease deficient host strain containing the lacIq repressor gene on an R-plasmid. The formation of active soluble alpha-glucosidase was almost 100% when E. coli cells induced in this manner were cultivated under conditions that reduced growth rate, i.e. at decreased temperature, extreme pH values or in minimal and complete media supplemented with different carbon sources.