title: Optimization of a heterologous signal peptide by site-directed mutagenesis for improved secretion of recombinant proteins in Escherichia coli.
abstract: A heterologous signal peptide (SP) from Bacillus sp. G1 was optimized for secretion of recombinant cyclodextrin glucanotransferase (CGTase) to the periplasmic and, eventually, extracellular space of Escherichia coli. Eight mutant SPs were constructed using site-directed mutagenesis to improve the secretion of recombinant CGTase. M5 is a mutated SP in which replacement of an isoleucine residue in the h-region to glycine created a helix-breaking or G-turn motif with decreased hydrophobicity. The mutant SP resulted in 110 and 94% increases in periplasmic and extracellular recombinant CGTase, respectively, compared to the wild-type SP at a similar level of cell lysis. The formation of intracellular inclusion bodies was also reduced, as determined by sodium dodecyl sulfate-polyacrylamyde gel electrophoresis, when this mutated SP was used. The addition of as low as 0.08% glycine at the beginning of cell growth improved cell viability of the E. coli host. Secretory production of other proteins, such as mannosidase, also showed similar improvement, as demonstrated by CGTase production, suggesting that the combination of an optimized SP and a suitable chemical additive leads to significant improvements of extracellular recombinant protein production and cell viability. These findings will be valuable for the extracellular production of recombinant proteins in E. coli.
authors.affiliation: Department of Bioprocess Engineering, Universiti Teknologi Malaysia, Skudai, Malaysia.
authors.forename: Mohd Anuar
authors.initials: MA
authors.lastname: Jonet
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: Raha Abdul
authors.initials: RA
authors.lastname: Rahim
authors.forename: Kheng Oon
authors.initials: KO
authors.lastname: Low
authors.forename: Rosli Md
authors.initials: RM
authors.lastname: Illias
mesh_terms: D001407:Bacillus / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D050505:Mutant Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase