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Enhanced expression of engineered ACA-less β-1, 6-N-acetylglucosaminidase (dispersin B) in Escherichia coli
- Source :
- Journal of Industrial Microbiology & Biotechnology. 36:1297-1305
- Publication Year :
- 2009
- Publisher :
- Oxford University Press (OUP), 2009.
-
Abstract
- beta-1,6-N-Acetylglucosaminidase (dispersin B), which cleaves poly-ss-(1,6)-linked N-acetylglucosamine, is encoded by dspB of Aggregatibacter actinomycetemcomitans. To enhance the production of dispersin B, we engineered dspB to transcribe mRNAs devoid of the trinucleotide ACA. Transcription and translation levels of ACA-less and wild-type dspB expressed in Escherichia coli (E. coli) under T5 and T7 promoters were analyzed by real-time RT-PCR and protein quantification, respectively. The ACA-less dspB mRNA level was significantly higher (P < 0.01) and produced 77.6 and 34.9% more dispersin B than wild-type dspB expressed under T7 and T5 promoters, respectively. Dispersin B expression under T7 promoter caused a 98-99.5% drop in the glyceraldehyde-3-phosphate dehydrogenase (gapA) mRNA level, which was not observed with T5 promoter. Fusion of green fluorescent protein (GFP) with dispersin B allowed rapid quantification of dispersin B production by measuring fluorescence intensity in culture broth. Although the cultures containing 0.1% glucose showed sustained increase in dispersin B-GFP production until 12 h, no significant increase in dispersin B activity was observed beyond 4 and 6 h after induction when expressed under T7 and T5 promoters, respectively. This study demonstrates the effectiveness of ACA-less mRNA and the advantage of GFP tagging for enhanced dispersin B production and quantification, which could be adapted for improving the production of other commercially important proteins in E. coli.
- Subjects :
- Glycoside Hydrolases
Transcription, Genetic
Green Fluorescent Proteins
Molecular Sequence Data
Bioengineering
Siphoviridae
Biology
medicine.disease_cause
Applied Microbiology and Biotechnology
Microbiology
Green fluorescent protein
Bacterial Proteins
Genes, Reporter
Transcription (biology)
Bacteriophage T7
Escherichia coli
medicine
Dispersin B
Promoter Regions, Genetic
Gene
Messenger RNA
Base Sequence
Nucleotides
Reverse Transcriptase Polymerase Chain Reaction
Gene Expression Profiling
Promoter
biology.organism_classification
Enterobacteriaceae
Molecular biology
Recombinant Proteins
Artificial Gene Fusion
Mutation
Biotechnology
Subjects
Details
- ISSN :
- 14765535 and 13675435
- Volume :
- 36
- Database :
- OpenAIRE
- Journal :
- Journal of Industrial Microbiology & Biotechnology
- Accession number :
- edsair.doi.dedup.....0d678a76d10ce4903befaf36baaf3333