Optimization of a heterologous signal peptide by site-directed mutagenesis for improved secretion of recombinant proteins in Escherichia coli

Mohd Anuar Jonet, Nor Muhammad Mahadi, Abdul Munir Abd. Murad, Amir Rabu, Farah Diba Abu Bakar, Raha Abdul Rahim, Kheng Oon Low, Rosli Md Illias

Research output: Contribution to journalArticle

35 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)48-58
Number of pages11
JournalJournal of Molecular Microbiology and Biotechnology
Volume22
Issue number1
DOIs
Publication statusPublished - Apr 2012

Fingerprint

Protein Sorting Signals
Site-Directed Mutagenesis
Recombinant Proteins
Escherichia coli
Glycine
Cell Survival
Mannosidases
Isoleucine
Inclusion Bodies
Extracellular Space
Hydrophobic and Hydrophilic Interactions
Sodium Dodecyl Sulfate
Bacillus
Electrophoresis
Gels
cyclomaltodextrin glucanotransferase
Growth
Proteins

Keywords

  • Cyclodextrin glucanotransferase
  • Escherichia coli
  • Heterologous signal peptide
  • Secretion
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology
  • Molecular Biology
  • Microbiology

Cite this

Optimization of a heterologous signal peptide by site-directed mutagenesis for improved secretion of recombinant proteins in Escherichia coli. / Jonet, Mohd Anuar; Mahadi, Nor Muhammad; Abd. Murad, Abdul Munir; Rabu, Amir; Abu Bakar, Farah Diba; Rahim, Raha Abdul; Low, Kheng Oon; Illias, Rosli Md.

In: Journal of Molecular Microbiology and Biotechnology, Vol. 22, No. 1, 04.2012, p. 48-58.

Research output: Contribution to journalArticle

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