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Application of an E. coli signal sequence as a versatile inclusion body tag
- Source :
- Microbial Cell Factories, 16(1):50. BioMed Central, Microbial Cell Factories, Jong, W S P, Vikström, D, Houben, D, Berg van Saparoea, H B, de Gier, J W & Luirink, J 2017, ' Application of an E. coli signal sequence as a versatile inclusion body tag ', Microbial Cell Factories, vol. 16, no. 1, 50 . https://doi.org/10.1186/s12934-017-0662-4
- Publication Year :
- 2017
- Publisher :
- BioMed Central, 2017.
-
Abstract
- Background Heterologous protein production in Escherichia coli often suffers from bottlenecks such as proteolytic degradation, complex purification procedures and toxicity towards the expression host. Production of proteins in an insoluble form in inclusion bodies (IBs) can alleviate these problems. Unfortunately, the propensity of heterologous proteins to form IBs is variable and difficult to predict. Hence, fusing the target protein to an aggregation prone polypeptide or IB-tag is a useful strategy to produce difficult-to-express proteins in an insoluble form. Results When screening for signal sequences that mediate optimal targeting of heterologous proteins to the periplasmic space of E. coli, we observed that fusion to the 39 amino acid signal sequence of E. coli TorA (ssTorA) did not promote targeting but rather directed high-level expression of the human proteins hEGF, Pla2 and IL-3 in IBs. Further analysis revealed that ssTorA even mediated IB formation of the highly soluble endogenous E. coli proteins TrxA and MBP. The ssTorA also induced aggregation when fused to the C-terminus of target proteins and appeared functional as IB-tag in E. coli K-12 as well as B strains. An additive effect on IB-formation was observed upon fusion of multiple ssTorA sequences in tandem, provoking almost complete aggregation of TrxA and MBP. The ssTorA-moiety was successfully used to produce the intrinsically unstable hEGF and the toxic fusion partner SymE, demonstrating its applicability as an IB-tag for difficult-to-express and toxic proteins. Conclusions We present proof-of-concept for the use of ssTorA as a small, versatile tag for robust E. coli-based expression of heterologous proteins in IBs. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0662-4) contains supplementary material, which is available to authorized users.
- Subjects :
- 0301 basic medicine
Signal peptide
Twin-arginine translocation pathway
Recombinant Fusion Proteins
Genetic Vectors
Heterologous
Bioengineering
Biology
Protein Sorting Signals
medicine.disease_cause
Applied Microbiology and Biotechnology
Inclusion bodies
03 medical and health sciences
Aggregation
Thioredoxins
Fusion tag
Protein biosynthesis
medicine
Escherichia coli
Humans
Heterologous protein production
Epidermal Growth Factor
Escherichia coli Proteins
Research
E. coli
Periplasmic space
Insolubility
SDG 10 - Reduced Inequalities
030104 developmental biology
Biochemistry
Solubility
Periplasmic Binding Proteins
Interleukin-3
Target protein
Carrier Proteins
Biotechnology
Subjects
Details
- Language :
- English
- ISSN :
- 14752859
- Volume :
- 16
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Microbial Cell Factories
- Accession number :
- edsair.doi.dedup.....1d5686686223213905d6d327b222e52a