NDLI: Enhanced expression of engineered ACA-less β-1, 6-N-acetylglucosaminidase (dispersin B) in Escherichia coli

Content Provider	Springer Nature Link
Author	Yakandawala, Nandadeva Gawande, Purushottam V. LoVetri, Karen Romeo, Tony Kaplan, Jeffrey B. Madhyastha, Srinivasa
Copyright Year	2009
Abstract	β-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.
Starting Page	1297
Ending Page	1305
Page Count	9
File Format	PDF
ISSN	13675435
Journal	Journal of Industrial Microbiology and Biotechnology
Volume Number	36
Issue Number	10
e-ISSN	14765535
Language	English
Publisher	Springer-Verlag
Publisher Date	2009-07-14
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Dispersin B ACA-less sequence MazEF GFP tagging Escherichia coli Microbiology Bioinformatics Biotechnology Genetic Engineering Inorganic Chemistry Biochemistry
Content Type	Text
Resource Type	Article
Subject	Medicine Bioengineering Applied Microbiology and Biotechnology Biotechnology

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