Open AccessSecretion of xylanase A2 in Streptomyces lividans : dependence on signal peptides length, number and composition
Author(s) -
Morosoli Rolf,
Dupont Claude
Publication year - 1999
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1999.tb08760.x
Subject(s) - signal peptide , signal peptidase , biochemistry , biology , gel electrophoresis , polyacrylamide gel electrophoresis , start codon , immunoprecipitation , sodium dodecyl sulfate , streptomyces , microbiology and biotechnology , peptide sequence , enzyme , gene , bacteria , messenger rna , genetics
The signal peptide (sp) in Streptomyces lividans xylanase A2 (XlnA2) was replaced by sps containing, in frame in their sequences, one, two, three or four initiation codons, each preceded by a Shine‐Dalgarno (SD) sequence. Precursors 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 dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) separation, precursors 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 these synthesized precursors was also shown to be inversely proportional to their length when comparing the specific activity of labelling versus sp length. In clones producing more than one precursor, a smear appeared on the autoradiograms, suggesting some degree of precursor degradation. As determined by pulse‐chase experiments, the rate of disappearance was almost the same for precursors of different lengths, but this might be the result of both true processing and proteolytic degradation. Furthermore, S. lividans rapidly degraded XlnA2 either when deprived of its sp or in the absence of the signal peptidase cleavage site.