ArticleMicrobial cell factories2015
Development of Pgrac100-based expression vectors allowing high protein production levels in Bacillus subtilis and relatively low basal expression in Escherichia coli.
Article in Microbial cell factories, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 29 papers.
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29 citing papers in PubMed, 65 citations in OpenAlex.
- Optimization Strategy of Expression Vectors and Regulatory Elements for Enhanced Protein Production inInternational journal of molecular sciences · 2025Review
- Engineering Bacillus subtilis for high-value bioproduction: recent advances and applications.Microbial cell factories · 2025Review
- Systematic modulation of bacterial resource allocation by perturbing RNA polymerase availability via synthetic transcriptional switches.Nucleic acids research · 2025Article
- 3,3-Bis(hydroxyaryl)oxindoles and Spirooxindoles Bearing a Xanthene Moiety: Synthesis, Mechanism, and Biological Activity.The Journal of organic chemistry · 2025Article
- Recombinant Production of Bovine αMicroorganisms · 2025Article
- Plasmid-encoded phosphatase RapP enhances cell growth in non-domesticated Bacillus subtilis strains.Nature communications · 2024Article
- Genome-Wide CRISPRi Screening of Key Genes for Recombinant Protein Expression in Bacillus Subtilis.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2024Article
- Non-consecutive enzyme interactions within TCA cycle supramolecular assembly regulate carbon-nitrogen metabolism.Nature communications · 2024Article
- Disruption of the bacterial OLE RNP complex impairs growth on alternative carbon sources.PNAS nexus · 2024Article
- A systematic analysis of affinity tags in the haloarchaeal expression system,Frontiers in microbiology · 2024Article
- A fitness trade-off between growth and survival governed by Spo0A-mediated proteome allocation constraints inScience advances · 2023Article
- Influence of N-terminal His-tags on the production of recombinant proteins in the cytoplasm ofBiotechnology reports (Amsterdam, Netherlands) · 2022Article
- Enhanced Extracellular Production and Characterization of Sucrose Isomerase inFoods (Basel, Switzerland) · 2022Article
- The multifunctionality of expression systems inExperimental biology and medicine (Maywood, N.J.) · 2021Review
- Cytosine Base Editor-Mediated Multiplex Genome Editing to Accelerate Discovery of Novel Antibiotics inFrontiers in microbiology · 2021Article
- Integrative expression vectors with PBiotechnology reports (Amsterdam, Netherlands) · 2020Article
- Pyruvate-responsive genetic circuits for dynamic control of central metabolism.Nature chemical biology · 2020Article
- Synthesis and In Vitro Cytotoxicity and Antibacterial Activity of Novel 1,2,3-Triazol-5-yl-phosphonates.Molecules (Basel, Switzerland) · 2020Article
- High copy number and highly stable Escherichia coli-Bacillus subtilis shuttle plasmids based on pWB980.Microbial cell factories · 2020Article
- Identification and characterization of sequence signatures in the Bacillus subtilis promoter PBiotechnology letters · 2020Article
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Authors and funding
4 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundIn general, fusion of recombinant genes to strong inducible promoters allowing intracellular expression in Bacillus subtilis is a two-step process. The ligation products are transformed into Escherichia coli, followed by identification of the correct plasmid, and this plasmid is subsequently transformed into B. subtilis. This raises the problem that basal level of expression of the recombinant gene could be harmful for E. coli cells. Based on the Pgrac promoter, we optimized the UP element, the -35, 15, -10 and the +1 region to enhance the promoter activity in B. subtilis after induction. However, detailed investigations for a promoter to develop expression vectors that allows high protein production levels in B. subtilis and a relatively low basal expression levels in E. coli has not been studied yet.
resultsWe screened the previously constructed library of E. coli - B. subtilis shuttle vectors for high level expression in B. subtilis and low basal level in E. coli. Promoter Pgrac100 turned out to meet these criteria, in which ß-galactosidase expression level of Pgrac100-bgaB is about 9.2 times higher than Pgrac01-bgaB in B. subtilis and the ratio of those in induced B. subtilis over un-induced E. coli from Pgrac100-bgaB is 1.3 times higher than Pgrac01-bgaB. Similarly, GFP expression level of Pgrac100-gfp is about 27 times higher than that of Pgrac01-gfp and the ratio from Pgrac100-gfp is 35.5 times higher than Pgrac01-gfp. This promoter was used as a basis for the construction of three novel vectors, pHT253 (His-tag-MCS), pHT254 (MCS-His-tag) and pHT255 (MCS-Strep-tag). Expression of the reporter proteins BgaB and GFP using these expression vectors in B. subtilis at a low IPTG concentration were measured and the fusion proteins could be purified easily in a single step by using Strep-Tactin or IMAC-Ni columns.
conclusionsThis paper describes the construction and analysis of an IPTG-inducible expression vector termed Pgrac100 for the high level production of intracellular recombinant proteins in B. subtilis and a relatively low basal expression level in E. coli. Based on this vector, the derivative vectors, Pgrac100-His-tag-MCS, Pgrac100-MCS-His-tag and Pgrac100-MCS-Strep-tag have been constructed.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.