| 1. |
- Jong, Wouter S. P., et al.
(författare)
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Application of an E. coli signal sequence as a versatile inclusion body tag
- 2017
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 16
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Tidskriftsartikel (refereegranskat)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.
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| 2. |
- Van Den Berg, F. D., et al.
(författare)
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In-line Characterisation of Microstructure and Mechanical Properties in the Manufacturing of Steel Strip for the Purpose of Product Uniformity Control
- 2016
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Ingår i: DGZfP-Proceedings BB 158. - 9783940283788
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Konferensbidrag (refereegranskat)abstract
- The uniformity of the microstructure of steel strip over the entire coil length and between different coils of the same grade is key to stable and consistent material behaviour in steel manufacturers’ proprietary processes, like rolling and levelling, and customers’ processes, like pressing and deep-drawing. In particular for high-strength steels, like dual phase and complex phase steels, the microstructure is very sensitive to processing variations resulting in a potentially larger spread in the mechanical properties of the product. In July 2013, a large European consortium consisting of 15 institutes started an RFCS [1] – funded project called “Product Uniformity Control” (PUC) with the primary objective to achieve enhanced and sustained product uniformity of steel strip by improved interpretation of data from inline measurement methods that aim 2 for real-time and non-destructive characterisation of microstructure and technomechanical parameters. Commonly, these techniques are based on electromagnetic (EM) or ultrasonic (US) measurement principles, which are favoured because of their non-destructive and potentially contact-free nature. To improve the techniques for inline materials characterisation, the PUC consortium takes a systematic approach to investigate the interrelations between mechanical properties -- microstructural parameters -- EM & US properties -- inline measurement thereof. The studies involve dedicated laboratory experiments, modelling of the EM and US properties of steel, modelling of inline measurement setups and statistical analysis of data from inline measurement systems. The synthesis of these activities should result in improved, model-based, calibrations and finally in a broader deployment and integration of the inline material characterisation techniques in steel manufacturing, adding value to the product and enhancing the process efficiency throughout the production chain from hot-rolling to finishing. This paper outlines the project approach, highlights interconnecting modelling and experimental research work, and demonstrates first results. Various contributions being presented at this WCNDT conference originate from the collaborative activities of this PUC project.
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| 3. |
- Wirdelius, Håkan, 1963, et al.
(författare)
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Validation of models for Laser Ultrasonic spectra as a function of the grain size in steel
- 2018
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Ingår i: 12th European Conference on Non-Destructive Testing (ECNDT 2018). - 9789163962172
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Konferensbidrag (refereegranskat)abstract
- To reduce costs of production and increase economic sustainability it is necessary to introduce quality assessment in an early stage in the manufacturing process. In an ongoing European project (Product Uniformity Control – PUC), the intention is to use ultrasonic information to assess microstructure parameters that are related to macroscale qualities such as mechanical properties. Laser induced ultrasonic technique (LUS) requires no media and can generate and detect ultrasonic information at some distance from the component. This technique is therefore addressed within this project as a solution to measure ultrasonic properties in an industrial environment. Mathematical modelling of the ultrasonic wave propagation problem has been used in order to get a deeper understanding of the physics and to identify ultrasonic properties that can be used as an indirect measurement of grain size. The use of both analytical and numerical models enabled extensive parametric studies together with investigation of ultrasonic interactions with well-defined individual microstructures. The LUS technique has previously been applied to e.g. monitor grain growth during thermomechanical processing of metals. These applications identified and used a correlation with the frequency content of the attenuation. This have been investigated as a possible indirect measurement of grain size, also in this project. The models have been used to verify the correlations and to evaluate different procedures that could be applied as an industrial solution. The suggested procedure is based on deconvolving two successive echoes and has been experimentally validated by two different LUS systems. The reference samples used in the validation were produced by changing the annealing temperature and time to obtain a variation in grain sizes. These grain sizes were then identified by EBSD and the samples were examined in terms of grain size influence on spectral attenuation
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