title: A mutant L-asparaginase II signal peptide improves the secretion of recombinant cyclodextrin glucanotransferase and the viability of Escherichia coli.
abstract: L-Asparaginase II signal peptide was used for the secretion of recombinant cyclodextrin glucanotransferase (CGTase) into the periplasmic space of E. coli. Despite its predominant localisation in the periplasm, CGTase activity was also detected in the extracellular medium, followed by cell lysis. Five mutant signal peptides were constructed to improve the periplasmic levels of CGTase. N1R3 is a mutated signal peptide with the number of positively charged amino acid residues in the n-region increased to a net charge of +5. This mutant peptide produced a 1.7-fold enhancement of CGTase activity in the periplasm and significantly decreased cell lysis to 7.8% of the wild-type level. The formation of intracellular inclusion bodies was also reduced when this mutated signal peptide was used as judged by SDS-PAGE. Therefore, these results provide evidence of a cost-effective means of expression of recombinant proteins in E. coli.
authors.affiliation: Department of Biology Sciences, Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.
authors.forename: Noor Faizah
authors.initials: NF
authors.lastname: Ismail
authors.forename: Salehhuddin
authors.initials: S
authors.lastname: Hamdan
authors.forename: Nor Muhammad
authors.initials: NM
authors.lastname: Mahadi
authors.forename: Abdul Munir Abdul
authors.initials: AM
authors.lastname: Murad
authors.forename: Amir
authors.initials: A
authors.lastname: Rabu
authors.forename: Farah Diba Abu
authors.initials: FD
authors.lastname: Bakar
authors.forename: Peter
authors.initials: P
authors.lastname: Klappa
authors.forename: Rosli Md
authors.initials: RM
authors.lastname: Illias
mesh_terms: D001215:Asparaginase / Q000235:genetics*; D003470:Culture Media / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D050296:Microbial Viability; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D019897:Periplasm / Q000201:enzymology; D021382:Protein Sorting Signals / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D003470:Culture Media; D050505:Mutant Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D001215:Asparaginase