I have cloned a gene containing signal peptide and cloned the same gene without signal peptide in expression vector pET-21 a(+). In E.coli BL21 codon plus both of the genes are expressed and secreted in Extracellular medium. Signal peptide is not cleaved as confirmed by N-terminal sequencing. Can any one suggest me some of its reasons?
Are you sure that the protein present in the extracellular medium is due to secretion and not to cell lysis ? ONe way to check would be to assay for a cytoplasmic marker, like betagalactosidase
Often when significantly over expressed the secretion machinery just can't process and cleave the signal peptide at the rate of production. I have frequently observed expression and secretion of a protein some of which is cleaved and some of which is not.
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