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J Ind Microbiol Biotechnol. 2009 Oct;36(10):1297-305. doi: 10.1007/s10295-009-0613-0. Epub 2009 Jul 14.

Enhanced expression of engineered ACA-less beta-1, 6-N-acetylglucosaminidase (dispersin B) in Escherichia coli.

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1
Kane Biotech Inc., Winnipeg, MB R3T 6C6, Canada.

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.

PMID:
19597857
DOI:
10.1007/s10295-009-0613-0
[Indexed for MEDLINE]

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