Experimental Evaluation of In Silico Selected Signal Peptides for Secretory Expression of Erwinia Asparaginase in Escherichia coli
Erwinia chrysanthemi asparaginase is an important drug used in cancer treatment, especially in acute lymphoblastic leukemia (ALL). Escherichia coli periplasmic space is an ideal compartment for recombinant expression of certain proteins. To efficient secretion, an appropriate signal sequence should be chosen for each protein individually. In this study, the proper signal sequences for secretory production of Erwinia asparaginase were predicted by in silico methods. Consequently, two signal peptides, OmpA and DsbA, were selected for secretory expression of the enzyme in E. coli. Asparaginase was translocated through the cytoplasmic membrane with either DsbA signal peptide or OmpA signal peptide; Using DsbA signal peptide, 5.95 units of the enzyme was obtained per milliliter of culture media, whereas OmpA signal sequence led to some amount of periplasmic expression. Our study showed that the co-translational signal peptide, DsbA, targeted the asparaginase to cell membrane more efficiently in comparison to the post-translational signal peptide, OmpA. The combination of in silico and experimental approaches provides a way to test a wide variety of signal sequences for secretory production of the enzyme in a time- and cost-effective manner. It is a fundamental step for further studies in the enzyme production process.
KeywordsErwinia asparaginase In silico Periplasmic space Secretion Signal peptide
The authors wish to thank Shiraz University of Medical Sciences for supporting the conduct of this research.
Compliance with Ethical Standards
Conflict of interest
All the authors declare that they have none conflict of interest.
Research Involving Human and Animal Participants
This article does not contain any studies with human participants or animals performed by any of the authors.
- Huber D, Boyd D, Xia Y, Olma MH, Gerstein M, Beckwith J (2005) Use of thioredoxin as a reporter to identify a subset of Escherichia coli signal sequences that promote signal recognition particle-dependent translocation. J Bacteriol 187:2983–2991. https://doi.org/10.1128/JB.187.9.2983-2991.2005 CrossRefPubMedPubMedCentralGoogle Scholar
- Low KO, Jonet MA, Ismail NF, Illias RM (2012) Optimization of a Bacillus sp signal peptide for improved recombinant protein secretion and cell viability in Escherichia coli: is there an optimal signal peptide design? Bioengineered 3:334–338. https://doi.org/10.4161/bioe.21454 CrossRefPubMedPubMedCentralGoogle Scholar
- Rau RE et al (2018) Outcome of pediatric patients with acute lymphoblastic leukemia/lymphoblastic lymphoma with hypersensitivity to pegaspargase treated with PEGylated Erwinia asparaginase, pegcrisantaspase: a report from the children's oncology group. Pediatric Blood Cancer 65:e26873. https://doi.org/10.1002/pbc.26873 CrossRefGoogle Scholar
- Samant S, Gupta G, Karthikeyan S, Haq SF, Nair A, Sambasivam G, Sukumaran S (2014) Effect of codon-optimized E. coli signal peptides on recombinant Bacillus stearothermophilus maltogenic amylase periplasmic localization, yield and activity. J Ind Microbiol Biotechnol 41:1435–1442. https://doi.org/10.1007/s10295-014-1482-8 CrossRefPubMedGoogle Scholar
- Zhang W, Lu J, Zhang S, Liu L, Pang X, Lv J (2018) Development an effective system to expression recombinant protein in E. coli via comparison and optimization of signal peptides: expression of Pseudomonas fluorescens BJ-10 thermostable lipase as case study. Microb Cell Fact 17: 50.CrossRefGoogle Scholar