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High-level expression of Bacillus naganoensis pullulanase from recombinant Escherichia coli with auto-induction: effect of lac operator.

Pullulanase plays an important role in specific hydrolysis of branch points in amylopectin and is generally employed as an important enzyme in starch-processing industry. So far, however, the production level of pullulanase is still somewhat low from wide-type strains and even heterologous expressio... Full description

Journal Title: PloS one 2013, Vol.8(10), p.e78416
Main Author: Nie, Yao
Other Authors: Yan, Wei , Xu, Yan , Chen, Wen Bo , Mu, Xiao Qing , Wang, Xinye , Xiao, Rong
Format: Electronic Article Electronic Article
Language: English
Subjects:
ID: E-ISSN: 1932-6203 ; DOI: 1932-6203 ; DOI: 10.1371/journal.pone.0078416
Link: http://search.proquest.com/docview/1449270890/?pq-origsite=primo
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title: High-level expression of Bacillus naganoensis pullulanase from recombinant Escherichia coli with auto-induction: effect of lac operator.
format: Article
creator:
  • Nie, Yao
  • Yan, Wei
  • Xu, Yan
  • Chen, Wen Bo
  • Mu, Xiao Qing
  • Wang, Xinye
  • Xiao, Rong
subjects:
  • Bacillus–Enzymology
  • Cloning, Molecular–Metabolism
  • Cryopreservation–Genetics
  • DNA Primers–Metabolism
  • DNA-Directed RNA Polymerases–Biosynthesis
  • Electrophoresis, Polyacrylamide Gel–Metabolism
  • Escherichia Coli–Genetics
  • Glycoside Hydrolases–Genetics
  • Lac Operon–Genetics
  • Lac Repressors–Metabolism
  • Models, Genetic–Genetics
  • Plasmids–Metabolism
  • Recombinant Proteins–Metabolism
  • Restriction Mapping–Metabolism
  • Transcriptional Activation–Metabolism
  • Viral Proteins–Metabolism
  • DNA Primers
  • Lac Repressors
  • Recombinant Proteins
  • Viral Proteins
  • Bacteriophage T7 RNA Polymerase
  • DNA-Directed RNA Polymerases
  • Glycoside Hydrolases
  • Pullulanase
ispartof: PloS one, 2013, Vol.8(10), p.e78416
description: Pullulanase plays an important role in specific hydrolysis of branch points in amylopectin and is generally employed as an important enzyme in starch-processing industry. So far, however, the production level of pullulanase is still somewhat low from wide-type strains and even heterologous expression systems. Here the gene encoding Bacillus naganoensis pullulanase was amplified and cloned. For expression of the protein, two recombinant systems, Escherichia coli BL21(DE3)/pET-20b(+)- pul and E. coli BL21(DE3)/pET-22b(+)- pul , were constructed, both bearing T7 promoter and signal peptide sequence, but different in the existance of lac operator and lacI gene encoding lac repressor. Recombinant pullulanase was initially expressed with the activity of up to 14 U/mL by E. coli BL21(DE3)/pET-20b(+)- pul with IPTG induction in LB medium, but its expression level reduced continually with the extension of cryopreservation time and basal expression was observed. However, E. coli BL21(DE3)/pET-22b(+)- pul , involving lac operator downstream of T7 promoter to regulate foreign gene transcription, exhibited pullulanase activity consistently without detected basal expression. By investigating the effect of lac operator, basal expression of foreign protein was found to cause expression instability and negative effect on production of target protein. Thus double-repression strategy was proposed that lac operators in both chromosome and plasmid were bound with lac repressor to repress T7 RNA polymerase synthesis and target protein expression before induction. Consequently, the total activity of pullulanase was remarkably increased to 580 U/mL with auto-induction by lac operator-involved E. coli BL21(DE3)/pET-22b(+)- pul . When adding 0.6% glycine in culture, the extracellular production of pullulanase was significantly improved with the extracellular activity of 502 U/mL, which is a relatively higher level achieved to date for extracellular production of pullulanase. The successful expression of pullulanase with lac operator regulation provides an efficient way for enhancement of expression stability and hence high-level production of target protein in recombinant E. coli .
language: eng
source:
identifier: E-ISSN: 1932-6203 ; DOI: 1932-6203 ; DOI: 10.1371/journal.pone.0078416
fulltext: fulltext
issn:
  • 19326203
  • 1932-6203
url: Link


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titleHigh-level expression of Bacillus naganoensis pullulanase from recombinant Escherichia coli with auto-induction: effect of lac operator.
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subjectBacillus–Enzymology ; Cloning, Molecular–Metabolism ; Cryopreservation–Genetics ; DNA Primers–Metabolism ; DNA-Directed RNA Polymerases–Biosynthesis ; Electrophoresis, Polyacrylamide Gel–Metabolism ; Escherichia Coli–Genetics ; Glycoside Hydrolases–Genetics ; Lac Operon–Genetics ; Lac Repressors–Metabolism ; Models, Genetic–Genetics ; Plasmids–Metabolism ; Recombinant Proteins–Metabolism ; Restriction Mapping–Metabolism ; Transcriptional Activation–Metabolism ; Viral Proteins–Metabolism ; DNA Primers ; Lac Repressors ; Recombinant Proteins ; Viral Proteins ; Bacteriophage T7 RNA Polymerase ; DNA-Directed RNA Polymerases ; Glycoside Hydrolases ; Pullulanase
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descriptionPullulanase plays an important role in specific hydrolysis of branch points in amylopectin and is generally employed as an important enzyme in starch-processing industry. So far, however, the production level of pullulanase is still somewhat low from wide-type strains and even heterologous expression systems. Here the gene encoding Bacillus naganoensis pullulanase was amplified and cloned. For expression of the protein, two recombinant systems, Escherichia coli BL21(DE3)/pET-20b(+)- pul and E. coli BL21(DE3)/pET-22b(+)- pul , were constructed, both bearing T7 promoter and signal peptide sequence, but different in the existance of lac operator and lacI gene encoding lac repressor. Recombinant pullulanase was initially expressed with the activity of up to 14 U/mL by E. coli BL21(DE3)/pET-20b(+)- pul with IPTG induction in LB medium, but its expression level reduced continually with the extension of cryopreservation time and basal expression was observed. However, E. coli BL21(DE3)/pET-22b(+)- pul , involving lac operator downstream of T7 promoter to regulate foreign gene transcription, exhibited pullulanase activity consistently without detected basal expression. By investigating the effect of lac operator, basal expression of foreign protein was found to cause expression instability and negative effect on production of target protein. Thus double-repression strategy was proposed that lac operators in both chromosome and plasmid were bound with lac repressor to repress T7 RNA polymerase synthesis and target protein expression before induction. Consequently, the total activity of pullulanase was remarkably increased to 580 U/mL with auto-induction by lac operator-involved E. coli BL21(DE3)/pET-22b(+)- pul . When adding 0.6% glycine in culture, the extracellular production of pullulanase was significantly improved with the extracellular activity of 502 U/mL, which is a relatively higher level achieved to date for extracellular production of pullulanase. The successful expression of pullulanase with lac operator regulation provides an efficient way for enhancement of expression stability and hence high-level production of target protein in recombinant E. coli .
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titleHigh-level expression of Bacillus naganoensis pullulanase from recombinant Escherichia coli with auto-induction: effect of lac operator.
authorNie, Yao ; Yan, Wei ; Xu, Yan ; Chen, Wen Bo ; Mu, Xiao Qing ; Wang, Xinye ; Xiao, Rong
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