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Design and production in Aspergillus niger of a chimeric protein associating a fungal feruloyl esterase and a clostridial dockerin domain

Author: Levasseur, A. · Pagès, S. · Fierobe, H.-P. · Navarro, D. · Punt, P. · Belaïch, J.-P. · Asther, M. · Record, E.
Type:article
Date:2004
Institution: TNO Voeding
Source:Applied and Environmental Microbiology, 12, 70, 6984-6991
Identifier: 238234
doi: doi:10.1128/AEM.70.12.6984-6991.2004
Keywords: Biology · Biotechnology · Amino acids · Bacteria · Bioassay · Electrophoresis · Enzymes · Fungi · Feruloyl esterase A (FAEA) · Fungal enzymes · Intracellular production · Western blotting · Proteins · antibody · bacterial protein · cell protein · chimeric protein · cohesin · esterase · fungal enzyme · glucan 1,4 alpha glucosidase · hybrid protein · protein dockerin · protein feruloyl esterase A · unclassified drug · enzyme · fungus · microbiology · protein · amino acid sequence · article · Aspergillus niger · cellular distribution · Clostridium thermocellum · complex formation · enzyme activity · enzyme binding · enzyme substrate complex · molecular weight · nonhuman · Northern blotting · polyacrylamide gel electrophoresis · posttranscriptional gene silencing · protein degradation · protein domain · protein processing · protein protein interaction · protein secretion · transcription initiation · Western blotting · Aspergillus niger · Bacterial Proteins · Biotechnology · Carboxylic Ester Hydrolases · Carrier Proteins · Cell Cycle Proteins · Chromosomal Proteins, Non-Histone · Clostridium thermocellum · Fungal Proteins · Nuclear Proteins · Recombinant Fusion Proteins · Transcription, Genetic · Transformation, Genetic · Actinobacteria (class) · Aspergillus · Aspergillus niger · Bacteria (microorganisms) · Clostridium · Clostridium thermocellum · Fungi · Posibacteria · uncultured actinomycete

Abstract

A chimeric enzyme associating feruloyl esterase A (FAEA) from Aspergilhis niger and dockerin from Clostridium thermocellum was produced in A. niger. A completely truncated form was produced when the dockerin domain was located downstream of the FAEA (FAEA-Doc), whereas no chimeric protein was produced when the bacterial dockerin domain was located upstream of the FAEA (Doc-FAEA). Northern blot analysis showed similar transcript levels for the two constructs, indicating a posttranscriptional bottleneck for Doc-FAEA production. The sequence encoding the first 514 amino acids from A. niger glucoamylase and a dibasic proteolytic processing site (kex-2) were fused upstream of the Doc-FAEA sequence. By using this fusion strategy, the esterase activity found in the extracellular medium was 20-fold-higher than that of the wild-type reference strain, and the production yield was estimated to be about 100 mg of chimeric protein/liter. Intracellular and extracellular production was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, dockerin-cohesin interaction assays, and Western blotting. Labeled cohesins detected an intact extracellular Doc-FAEA of about 43 kDa and a cleaved-off dockerin domain of about 8 kDa. In addition, an intracellular 120-kDa protein was recognized by using labeled cohesins and antibodies raised against FAEA. This protein corresponded to the unprocessed Doc-FAEA form fused to glucoamylase. In conclusion, these results indicated that translational fusion to glucoamylase improved the secretion efficiency of a chimeric Doc-FAEA protein and allowed production of the first functional fungal enzyme joined to a bacterial dockerin.