Skip to main content
SpringerLink
Log in

A mutant l-asparaginase II signal peptide improves the secretion of recombinant cyclodextrin glucanotransferase and the viability of Escherichia coli

  • Original Research Paper
  • Published:
Biotechnology Letters Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Akita M, Sasaki S, Matsuyama S, Mizushima S (1990) SecA interacts with secretory proteins by recognizing the positive charge at the amino terminus of the signal peptide in Escherichia coli. J Biol Chem 265:8164–8169

    PubMed  CAS  Google Scholar 

  • Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421

    Article  PubMed  CAS  Google Scholar 

  • Chen H, Kim J, Kendall DA (1996) Competition between functional signal peptides demonstrates variation in affinity for the secretion pathway. J Bacteriol 178:6658–6664

    PubMed  CAS  Google Scholar 

  • Chou MM, Kendall DA (1990) Polymeric sequences reveal a functional interrelationship between hydrophobicity and length of signal peptides. J Biochem 265:2873–2880

    CAS  Google Scholar 

  • Fang N, Zhong CQ, Liang X, Tang XF, Tang B (2010) Improvement of extracellular production of a thermophilic subtilase expressed in Escherichia coli by random mutagenesis of its N-terminal propeptide. Appl Microbiol Biotechnol 85:1473–1481

    Article  PubMed  CAS  Google Scholar 

  • Fu ZB, Ng KL, Lam TL, Wong WKR (2005) Cell death caused by hyper-expression of a secretory exoglucanase in Escherichia coli. Protein Expr Purif 42:67–77

    Article  PubMed  CAS  Google Scholar 

  • Gennity J, Goldstein J, Inouye M (1990) Signal peptide mutants of Escherichia coli. J Bioenerg Biomembr 22:233–269

    Article  PubMed  CAS  Google Scholar 

  • Jemli S, Messaoud E, Mabrouk S, Bejar S (2008) The cyclodextrin glycosyltransferase of Paenibacillus pabuli US132 strain: molecular characterization and overproduction of the recombinant enzyme. J Biomed Biotechnol. doi:10.1155/2008/692573

  • Kim MH, Lee JK, Kim HK, Sohn CB, Oh TK (1999) Overexpression of cyclodextrin glycosyltransferase gene from Brevibacillus brevis in Escherichia coli by control of temperature and mannitol concentration. Biotechnol Tech 13:765–770

    Article  CAS  Google Scholar 

  • Matsumi R, Atomi H, Imanaka T (2005) Biochemical properties of a putative signal peptide peptidase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. J Bacteriol 187:7072–7080

    Article  PubMed  CAS  Google Scholar 

  • Nesmeyanova MA, Karamyshev AL, Karamysheva ZN, Kalinin AE, Ksenzenko VN, Kajava AV (1997) Positively charged lysine at the N-terminus of the signal peptide of the Escherichia coli alkaline phosphatase provides the secretion efficiency and is involved in the interaction with anionic phospholipids. Fed Eur Biochem Soc 403:203–207

    CAS  Google Scholar 

  • Ni Y, Chen R (2009) Extracellular recombinant protein production from Escherichia coli. Biotechnol Lett 31:1661–1670

    Article  PubMed  CAS  Google Scholar 

  • Ong RM, Goh KM, Mahadi NM, Hassan O, Rahman RN, Illias RM (2008) Cloning, extracellular expression and characterization of a predominant beta-CGTase from Bacillus sp. G1 in E. coli. J Ind Microbiol Biotechnol 35:1705–1714

    Article  PubMed  CAS  Google Scholar 

  • Pugsley AP, Francetic O (1998) Protein secretion in Escherichia coli K-12: dead or alive? Cell Mol Life Sci 54:347–352

    Article  PubMed  CAS  Google Scholar 

  • Shin HD, Chen RR (2008) Extracellular recombinant protein production from an Escherichia coli lpp deletion mutant. Biotechnol Bioeng 101:1288–1296

    Article  PubMed  CAS  Google Scholar 

  • Shokri A, Sandén AM, Larsson G (2002) Growth rate-dependent changes in Escherichia coli membrane structure and protein leakage. Appl Microbiol Biotechnol 58:386–392

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This research was funded under the Genomics and Molecular Biology Initiatives Programme of the Malaysia Genome Institute, Ministry of Science, Technology and Innovation Malaysia (Project No. 07-05-MGI-GMB011).

Author information

Authors and Affiliations

  1. Department of Biology Sciences, Faculty of Biosciences and Bioengineering, Universiti Teknologi Malaysia, 81310, Skudai, Johor, Malaysia

    Noor Faizah Ismail & Salehhuddin Hamdan

  2. Malaysia Genome Institute, UKM-MTDC, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia

    Nor Muhammad Mahadi

  3. School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia

    Abdul Munir Abdul Murad, Amir Rabu & Farah Diba Abu Bakar

  4. School of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK

    Peter Klappa

  5. Department of Bioprocess Engineering, Faculty of Chemical Engineering, Universiti Teknologi Malaysia, 81310, Skudai, Johor, Malaysia

    Noor Faizah Ismail, Salehhuddin Hamdan & Rosli Md. Illias

Authors
  1. Noor Faizah Ismail
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Salehhuddin Hamdan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nor Muhammad Mahadi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Abdul Munir Abdul Murad
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Amir Rabu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Farah Diba Abu Bakar
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Peter Klappa
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Rosli Md. Illias
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rosli Md. Illias.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ismail, N.F., Hamdan, S., Mahadi, N.M. et al. A mutant l-asparaginase II signal peptide improves the secretion of recombinant cyclodextrin glucanotransferase and the viability of Escherichia coli . Biotechnol Lett 33, 999–1005 (2011). https://doi.org/10.1007/s10529-011-0517-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-011-0517-8

Keywords

Search

Navigation