ArticleApplied and environmental microbiology2000
Compatible-solute-supported periplasmic expression of functional recombinant proteins under stress conditions.
Article in Applied and environmental microbiology, 2000. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 47 papers.
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Who cites it
47 citing papers in PubMed, 178 citations in OpenAlex.
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- Structural characterization of a novel cyclic 2,3-diphosphoglycerate synthetase involved in extremolyte production in the archaeonFrontiers in microbiology · 2023Article
- A recombinant Der p 1-specific allergen-toxin demonstrates superior killing of allergen-reactive IgGImmunotherapy advances · 2023Article
- Enhanced Recombinant Protein Production Under Special Environmental Stress.Frontiers in microbiology · 2021Review
- The saltern-derived3 Biotech · 2020Article
- The architecture of the diaminobutyrate acetyltransferase active site provides mechanistic insight into the biosynthesis of the chemical chaperone ectoine.The Journal of biological chemistry · 2020Article
- Two MarR-Type Repressors Balance Precursor Uptake and Glycine Betaine Synthesis inFrontiers in microbiology · 2020Article
- Exploiting Substrate Promiscuity of Ectoine Hydroxylase for Regio- and Stereoselective Modification of Homoectoine.Frontiers in microbiology · 2019Article
- Biosynthesis of the Stress-Protectant and Chemical Chaperon Ectoine: Biochemistry of the Transaminase EctB.Frontiers in microbiology · 2019Article
- Uncovering the Mechanisms of Halotolerance in the Extremely Acidophilic Members of theFrontiers in microbiology · 2019Article
- Stabilization of dry protein coatings with compatible solutes.Biointerphases · 2018Article
- Review
- Tinkering with Osmotically Controlled Transcription Allows Enhanced Production and Excretion of Ectoine and Hydroxyectoine from a Microbial Cell Factory.Applied and environmental microbiology · 2018Article
- Transcriptomic and Ectoine Analysis of HalotolerantFrontiers in microbiology · 2018Article
- Effects of Ectoine on Behavior and Candidate Genes Expression in ICV-STZ Rat Model of Sporadic Alzheimer's Disease.Advanced pharmaceutical bulletin · 2017Article
- Efficient targeting of CD13 on cancer cells by the immunotoxin scFv13-ETA' and the bispecific scFv [13xds16].Journal of cancer research and clinical oncology · 2017Article
- Improvement in the production of the human recombinant enzyme N-acetylgalactosamine-6-sulfatase (rhGALNS) in Escherichia coli using synthetic biology approaches.Scientific reports · 2017Article
- Fully human MAP-fusion protein selectively targets and eliminates proliferating CD64(+) M1 macrophages.Immunology and cell biology · 2016Article
- Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen.BMC biotechnology · 2016Article
- Effects of Environmental Factors on Soluble Expression of a Humanized Anti-TNF-α scFv Antibody in Escherichia coli.Advanced pharmaceutical bulletin · 2015Article
Corrections and comments
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Authors and funding
6 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The standard method of producing recombinant proteins such as immunotoxins (rITs) in large quantities is to transform gram-negative bacteria and subsequently recover the desired protein from inclusion bodies by intensive de- and renaturing procedures. The major disadvantage of this technique is the low yield of active protein. Here we report the development of a novel strategy for the expression of functional rIT directed to the periplasmic space of Escherichia coli. rITs were recovered by freeze-thawing of pellets from shaking cultures of bacteria grown under osmotic stress (4% NaCl plus 0.5 M sorbitol) in the presence of compatible solutes. Compatible solutes, such as glycine betaine and hydroxyectoine, are low-molecular-weight osmolytes that occur naturally in halophilic bacteria and are known to protect proteins at high salt concentrations. Adding 10 mM glycine betaine for the cultivation of E. coli under osmotic stress not only allowed the bacteria to grow under these otherwise inhibitory conditions but also produced a periplasmic microenvironment for the generation of high concentrations of correctly folded rITs. Protein purified by combinations of metal ion affinity and size exclusion chromatography was substantially stabilized in the presence of 1 M hydroxyecotine after several rounds of freeze-thawing, even at very low protein concentrations. The binding properties and cytotoxic potency of the rITs were confirmed by competitive experiments. This novel compatible-solute-guided expression and purification strategy might also be applicable for high-yield periplasmic production of recombinant proteins in different expression systems.
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