Secretion of xylanase A2 in Streptomyces lividans: dependence on signal peptides length, number and composition

The signal peptide (sp) in Streptomyces lividans xylanase A2 (XlnA2) was re placed by sps containing, in frame in their sequences, one, two, three or f our initiation codons, each preceded by a Shine-Dalgarno (SD) sequence. Pre cursors of the corresponding proteins should thus have sps of, respectively , 27, 46, 82 and 91 amino acids (aa) long. By radiolabelling of S. lividans harboring the different constructs inserted in a multicopy plasmid and by immunoprecipitation with anti-xylanase antibodies followed by sodium dodecy l sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation, precurs ors of the expected sizes were obtained in each clone. This indicates that ribosomes can synthesize different XlnA2 precursors from initiation codons inserted in the sp sequence, independently of their number. The amount of t hese synthesized precursors was also shown to be inversely proportional to their length when comparing the specific activity of labelling versus sp le ngth. In clones producing more than one precursor, a smear appeared on the autoradiograms, suggesting some degree of precursor degradation. As determi ned by pulse-chase experiments, the rate of disappearance was almost the sa me for precursors of different lengths, but this might be the result of bot h true processing and proteolytic degradation. Furthermore, S. lividans rap idly degraded XlnA2 either when deprived of its sp or in the absence of the signal peptidase cleavage site. (C) 1999 Federation of European Microbiolo gical Societies. Published by Elsevier Science B.V. Ali rights reserved.