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A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger

  • Applied Genetics and Molecular Biotechnology
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Abstract

A set of different integrative expression vectors for the intracellular production of recombinant proteins with or without affinity tag in Aspergillus niger was developed. Target genes can be expressed under the control of the highly efficient, constitutive pkiA promoter or the novel sucrose-inducible promoter of the β-fructofuranosidase (sucA) gene of A. niger in the presence or absence of alternative carbon sources. All expression plasmids contain an identical multiple cloning sequence that allows parallel construction of N- or C-terminally His6- and StrepII-tagged versions of the target proteins. Production of two heterologous model proteins, the green fluorescence protein and the Thermobifida fusca hydrolase, proved the functionality of the vector system. Efficient production and easy detection of the target proteins as well as their fast purification by a one-step affinity chromatography, using the His6- or StrepII-tag sequence, was demonstrated.

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Acknowledgements

We thank Drs. Jana Melzer, Martin Kucklick, and Martin Reisinger for discussions and critical reading of the manuscript and Susanne Kneip, Alga Zuccaro, and Arne Homann for helpful advice and valuable technical assistance. We also thank Reginald Storms and Yang Yang for plasmids. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB578, TP A1) and the Fonds der Deutschen Chemie.

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Authors and Affiliations

  1. Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, 38106, Braunschweig, Germany

    Andreas H. F. J. Roth & Petra Dersch

  2. Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse. 7, 38124, Braunschweig, Germany

    Petra Dersch

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  1. Andreas H. F. J. Roth
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  2. Petra Dersch
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Correspondence to Petra Dersch.

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Fig. S1

P sucA -driven GFP expression prior and after induction with sucrose. A. niger strains ARAn17, ARAn55, ARAn56, and ARAn101 (P sucA ::gfp) were cultivated for 48 h in liquid minimal medium under standard conditions with 100 mM glucose prior to the induction with 25 mM sucrose. After induction, samples were taken at indicated time points, and total RNA was prepared. Total RNA of the samples were separated, and Northern analysis was performed with a GFP-specific probe. The sizes of the RNA marker are indicated on the left, and 18S rRNA of the loaded samples are shown below. (JPG 566 kb)

Table S1

Oligonucleotides used in this study. (DOC 53 kb)

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Roth, A.H.F.J., Dersch, P. A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger . Appl Microbiol Biotechnol 86, 659–670 (2010). https://doi.org/10.1007/s00253-009-2252-9

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