Enhanced expression of engineered ACA-less beta-1, 6-N-acetylglucosaminidase (dispersin B) in Escherichia coli.
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.
Yakandawala
Nandadeva
N
Kane Biotech Inc., Winnipeg, MB R3T 6C6, Canada.
Gawande
Purushottam V
PV
LoVetri
Karen
K
Romeo
Tony
T
Kaplan
Jeffrey B
JB
Madhyastha
Srinivasa
S
D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics*; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017906:Siphoviridae / Q000235:genetics; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D009711:Nucleotides; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D006026:Glycoside Hydrolases; C501330:dispersin B, Actinobacillus actinomycetemcomitans