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Living Modified Organism (LMO)
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Barley modified for the production of LL-37 peptide
EN
bHOR:LL-37
No
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Olomouc,Person:Palacky University Olomouc, Institute of Molecular and Translational Medicine
77900, Czech RepublicPhone: +420 585 632 111,Fax:Email: reception@imtm.upol.cz,Related OrganizationPalacky University Olomouc ()Academic or research institute
The barley (Hordeum vulgare) was modified for the production of human LL-37 peptide, which has known broad spectrum antimicrobial activity and acts as component of the basal immune response to infection. Barley production platforms have little phenolic compound content and have low amount of proteolytic enzymatic activity.
The line is one of three lines (see "Related LMOs") being tested for bioproduction and protein purification. This line contains an endosperm specific promoter and no protein purification tag sequences. All lines demonstrated normal phenotypes and the LL-37 peptide was shown to be bioactive.
The modified barley additionally contains an antibiotic selection marker, Escherichia coli hygromycin phosphotransferase B, for hygromycin selection during transformation.
EN
The line is one of three lines (see "Related LMOs") being tested for bioproduction and protein purification. This line contains an endosperm specific promoter and no protein purification tag sequences. All lines demonstrated normal phenotypes and the LL-37 peptide was shown to be bioactive.
The modified barley additionally contains an antibiotic selection marker, Escherichia coli hygromycin phosphotransferase B, for hygromycin selection during transformation.
The term “Recipient organism” refers to an organism (either already modified or non-modified) that was subjected to genetic modification, whereas “Parental organisms” refers to those that were involved in cross breeding or cell fusion.
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BCH-ORGA-SCBD-12110-5 Organism Hordeum vulgare (Barley, HORVU)Crops
Hordeum vulgare cultivar Golden promise
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Barley modified for the production of LL-37 peptide| Palacky University Olomouc | Production of medical or pharmaceutical compounds (human or animal), Resistance to antibiotics (Hygromycin), Selectable marker genes and reporter genes
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Barley modified for the production of LL-37 peptide| Palacky University Olomouc | Production of medical or pharmaceutical compounds (human or animal), Resistance to antibiotics (Hygromycin), Selectable marker genes and reporter genes
pBRACT209
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- Agrobacterium-mediated DNA transfer
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Some of these genetic elements may be present as fragments or truncated forms. Please see notes below, where applicable.
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BCH-GENE-SCBD-115696-1 LL-37 peptide | Homo sapiens (HUMAN)Protein coding sequence | Production of medical or pharmaceutical compounds (human or animal)
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BCH-GENE-SCBD-14991-8 Hygromycin B phosphotransferase gene | Escherichia coli (ECOLX)Protein coding sequence | Resistance to antibiotics (Hygromycin),Selectable marker genes and reporter genes
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BCH-GENE-SCBD-103023-2 KDEL ER retention signalTransit signal
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BCH-GENE-SCBD-100269-8 Nopaline Synthase Gene Terminator | Agrobacterium tumefaciens (Agrobacterium)Terminator
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BCH-GENE-SCBD-115697-1 Cytokinin dehydrogenase 1 signal peptide | Zea mays (Maize, Corn, MAIZE)Transit signal
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BCH-GENE-SCBD-100287-7 CaMV 35S promoter | Cauliflower mosaic virus (CaMV)Promoter
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BCH-GENE-SCBD-101595-2 Hordein B1 promoter | Hordeum vulgare (Barley, HORVU)Promoter
Gene cassettes
The DNA insertion contain the following two gene cassettes:
1) Escherichia coli hygromycin B phosphotransferase (hph); and
2) Homo sapiens LL-37 peptide.
Gene expression
Transcription of hph is under control of the Cauliflower mosaic virus 35S promoter and the Agrobacterium tumefaciens nopaline synthase (nos) terminator. Due to the nature of the viral promoter, transcription is expected to occur at high levels.
Transcription of the human LL-37 peptide occurs from the Hordeum vulgare hordein B1 (bHOR) promoter and terminates at the nos terminator. The transcript contains the following (from 5' to 3'): Zea mays cytokinin dehydrogenase 1 (ZmCKX1sp) signal peptide, LL-37 peptide and a synthetic KDEL ER retention signal. The bHOR promoter restricts expression to the barley endosperm. The synthetic eukaryotic KDEL sequence facilitates the transport of LL-37 peptide to the endoplasmic reticulum (ER). Prior to export from the ER, the KDEL sequence is cleaved from the peptide. ZmCKX1sp facilitates the transit of LL-37 through the ER and excretion into the apoplast.
Note
- Agrobacterium tumefaciens strain AG1 was used in the transformation of barley immature zygotic embryos.
- The bHOR promoter sequence has the following accession number GenBank: X87232.1.
- The final LL-37 peptide is not expected to retain the signal peptides.
EN
The DNA insertion contain the following two gene cassettes:
1) Escherichia coli hygromycin B phosphotransferase (hph); and
2) Homo sapiens LL-37 peptide.
Gene expression
Transcription of hph is under control of the Cauliflower mosaic virus 35S promoter and the Agrobacterium tumefaciens nopaline synthase (nos) terminator. Due to the nature of the viral promoter, transcription is expected to occur at high levels.
Transcription of the human LL-37 peptide occurs from the Hordeum vulgare hordein B1 (bHOR) promoter and terminates at the nos terminator. The transcript contains the following (from 5' to 3'): Zea mays cytokinin dehydrogenase 1 (ZmCKX1sp) signal peptide, LL-37 peptide and a synthetic KDEL ER retention signal. The bHOR promoter restricts expression to the barley endosperm. The synthetic eukaryotic KDEL sequence facilitates the transport of LL-37 peptide to the endoplasmic reticulum (ER). Prior to export from the ER, the KDEL sequence is cleaved from the peptide. ZmCKX1sp facilitates the transit of LL-37 through the ER and excretion into the apoplast.
Note
- Agrobacterium tumefaciens strain AG1 was used in the transformation of barley immature zygotic embryos.
- The bHOR promoter sequence has the following accession number GenBank: X87232.1.
- The final LL-37 peptide is not expected to retain the signal peptides.
EN
- Pharmaceutical
- Research
The LL-37 peptide was only detected in the grains of the modified barley and not the roots or leaves (see "Molecular Farming in Barley: Development of a Novel Production Platform to Produce Human Antimicrobial Peptide LL-37").
EN
Cathelicidin antimicrobial peptide is the only cathelicidin protein found in humans and is location on chromosome 3p21. The sequence contains 4 exons and is translated to hCAP18, a pre-pro-protein, containing signal peptide, a conserved pro-sequence (cathelin-like domain) and a C-terminal antimicrobial peptide, LL-37. The active LL-37 peptide is produced from proteolytic cleavage from hCAP18 and its primary structure is based on 37 amino acid residues (~ 18kDa), which form an amiphiphatic alpha-helix (secondary) structure.
EN
- Molecular Farming in Barley Development of a NovelProduction Platform to Produce Human AntimicrobialPeptide LL-37.pdf [ English ]
- John Innes Centre - Crop transformation (BRACT vectors) [ English ]
- pBract209 sequence map.doc [ English ]
- EU Joint Research Centre - Deliberate Release into the Environment and Placing on the EU Markets of GMO - GMO Register [ English ]
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