Abstract: Various tags, especially charged amino acids, were widely applied in soluble expression and secretion of heterogenous proteins in Escherichia coli recent years. To know how the charged amino acids types and numbers influent the Pul13A expression and secretion, 25 Pul13A mutants were constructed, which had different N-terminal amino acid tags. Among them, the nine-glutamine tag could decrease the transcription level and increase the N-terminal mRNA stability, and Pul13A′s activity changed to 179.60 U/mL that was 278.28% higher than the control without any amino acid tag. Meanwhile, the N-terminal seven-aspartate tag enhanced the inner membrane permeability, and secreted massive pullulanse into the culture medium ( 48.18 U/mL ). Furthermore, it was found that the CBM41 structural domain was different in different mutants, which maybe mainly result in the differences of the expression and secretion levels.

Key words: charged-amino-acid tags, expression and secretory, mRNA, membrane permeability, protein structure prediction