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.
Similar content being viewed by others
References
Bakri Y, Al-Jazairi M, Al-Kayat G (2008) Xylanase production by a newly isolated Aspergillus niger SS7 in submerged culture. Pol J Microbiol 57:249–251
Boddy LM, Berges T, Barreau C, Vainstein MH, Dobson MJ, Ballance DJ, Peberdy JF (1993) Purification and characterisation of an Aspergillus niger invertase and its DNA sequence. Curr Genet 24:60–66
Bohlin C, Jönsson LJ, Roth R, van Zyl WH (2006) Heterologous expression of Trametes versicolor laccase in Pichia pastoris and Aspergillus niger. Appl Biochem Biotechnol 129:195–214
Bos CJ, Debets AJ, Swart K, Huybers A, Kobus G, Slakhorst SM (1988) Genetic analysis and the construction of master strains for assignment of genes to six linkage groups in Aspergillus niger. Curr Genet 14:437–443
Chill L, Trinh L, Azadi P, Ishihara M, Sonon R, Karnaukhova E, Ophir Y, Golding B, Shiloach J (2009) Production, purification, and characterization of human α1 proteinase inhibitor from Aspergillus niger. Biotechnol Bioeng 102:828–844
Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien RY (1995) Understanding, improving and using green fluorescent proteins. Trends Biochem Sci 20:448–455
Davies RW (1994) Heterologous gene expression and protein secretion in Aspergillus. Prog Ind Microbiol 29:527–560
de Graaff L, van den Broeck H, Visser J (1992) Isolation and characterization of the Aspergillus niger pyruvate kinase gene. Curr Genet 22:21–27
Dresler K, van den Heuvel J, Müller RJ, Deckwer WD (2006) Production of a recombinant polyester-cleaving hydrolase from Thermobifida fusca in Escherichia coli. Bioprocess Biosyst Eng 29:169–183
Fowler T, Berka RM, Ward M (1990) Regulation of the glaA gene of Aspergillus niger. Curr Genet 18:537–545
Ganzlin M, Rinas U (2008) In-depth analysis of the Aspergillus niger glucoamylase (glaA) promoter performance using high-throughput screening and controlled bioreactor cultivation techniques. J Biotechnol 135:266–271
Gordon CL, Archer DB, Jeenes DJ, Doonan JH, Wells B, Trinci AP, Robson GD (2000) A glucoamylase::GFP gene fusion to study protein secretion by individual hyphae of Aspergillus niger. J Microbiol Methods 42:39–48
Grote A, Hiller K, Scheer M, Munch R, Nortemann B, Hempel DC, Jahn D (2005) JCat: a novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Res 33:W526–W531
Heim R, Cubitt AB, Tsien RY (1995) Improved green fluorescence. Nature 373:663–664
Karnaukhova E, Ophir Y, Trinh L, Dalal N, Punt PJ, Golding B, Shiloach J (2007) Expression of human α1-proteinase inhibitor in Aspergillus niger. Microb Cell Fact 6:34
Krasevec N, van de Hondel CA, Komel R (2000) Expression of human lymphotoxin-α in Aspergillus niger. Pflugers Arch 440:R83–R85
Lee SB, Milgroom MG, Tayler JW (1988) A rapid, high-yield mini-prep method for isolation of total genomic DNA from fungi. Fungal Genet Newsl 35:23–24
Lichty JJ, Malecki JL, Agnew HD, Michelson-Horowitz DJ, Tan S (2005) Comparison of affinity tags for protein purification. Protein Expr Purif 41:98–105
Mattern IE, van Noort JM, van den Berg P, Archer DB, Roberts IN, van den Hondel CA (1992) Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases. Mol Gen Genet 234:332–336
Pontecorvo G, Roper JA, Forbes E (1953) Genetic recombination without sexual reproduction in Aspergillus niger. J Gen Microbiol 8:198–210
Porath J, Carlsson J, Olsson I, Belfrage G (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258:598–599
Punt PJ, van den Hondel CA (1992) Transformation of filamentous fungi based on hygromycin B and phleomycin resistance markers. Methods Enzymol 216:447–457
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. CSHL, Cold Spring Harbor
Schmidt TG, Koepke J, Frank R, Skerra A (1996) Molecular interaction between the Strep-tag affinity peptide and its cognate target, streptavidin. J Mol Biol 255:753–766
Shah P, Bhavsar K, Soni SK, Khire JM (2009) Strain improvement and up scaling of phytase production by Aspergillus niger NCIM 563 under submerged fermentation conditions. J Ind Microbiol Biotechnol 36:373–380
Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59:223–239
Storms R, Zheng Y, Li H, Sillaots S, Martinez-Perez A, Tsang A (2005) Plasmid vectors for protein production, gene expression and molecular manipulations in Aspergillus niger. Plasmid 53:191–204
van Hartingsveldt W, Mattern IE, van Zeijl CM, Pouwels PH, van den Hondel CA (1987) Development of a homologous transformation system for Aspergillus niger based on the pyrG gene. Mol Gen Genet 206:71–75
Verdoes JC, Punt PJ, van der Berg P, Debets F, Stouthamer AH, van den Hondel CA (1994) Characterization of an efficient gene cloning strategy for Aspergillus niger based on an autonomously replicating plasmid: cloning of the nicB gene of A. niger. Gene 146:159–165
Vishniac W, Santer M (1957) The thiobacilli. Bacteriol Rev 21:195–213
Ward M, Lin C, Victoria DC, Fox BP, Fox JA, Wong DL, Meerman HJ, Pucci JP, Fong RB, Heng MH, Tsurushita N, Gieswein C, Park M, Wang H (2004) Characterization of humanized antibodies secreted by Aspergillus niger. Appl Environ Microbiol 70:2567–2576
Wiebe MG, Karandikar A, Robson GD, Trinci AP, Candia JL, Trappe S, Wallis G, Rinas U, Derkx PM, Madrid SM, Sisniega H, Faus I, Montijn R, van den Hondel CA, Punt PJ (2001) Production of tissue plasminogen activator (t-PA) in Aspergillus niger. Biotechnol Bioeng 76:164–174
Yang Y, Malten M, Grote A, Jahn D, Deckwer WD (2007) Codon optimized Thermobifida fusca hydrolase secreted by Bacillus megaterium. Biotechnol Bioeng 96:780–794
Zuccaro A, Götze S, Kneip S, Dersch P, Seibel J (2008) Tailor-made fructooligosaccharides by a combination of substrate and genetic engineering. Chembiochem 9:143–149
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.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
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)
Rights and permissions
About this article
Cite this article
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
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-009-2252-9