Barley modified for the production of LL-37 peptide | BCH-LMO-SCBD-115700 | Living Modified Organism | Biosafety Clearing-House

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Living Modified Organism (LMO)

Decisions on the LMO Risk Assessments  
last updated: 01 Sep 2020
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Barley modified for the production of LL-37 peptide
EN
bHOR:MBP_LL-37
No
The barley 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 protein purification tag sequences (hexahistidine and maltose binding protein), which can be removed post-purification using an enterokinase. The hexahistidine tag allows for protein purification using immobilized metal affinity chromatography, while the maltose binding protein improves the solubility and folding of the recombinant LL-37. 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 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.
Hordeum vulgare cultivar Golden promise
EN
  • 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
  • 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
Characteristics of the modification process
pBRACT209
EN
  • Agrobacterium-mediated DNA transfer
Some of these genetic elements may be present as fragments or truncated forms. Please see notes below, where applicable.
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'): a Zea mays cytokinin dehydrogenase 1 (ZmCKX1sp) signal peptide, a synthetic hexahistidine affinity tag, an E. coli maltose binding protein affinity tag, the 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. Prior to export from the ER, the translated peptide is cleaved from the peptide. ZmCKX1sp facilitates the transit of LL-37 through the ER and excretion from the cell. The final protein will retain the hexahistidine and maltose binding protein tags for purification.

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
LMO characteristics
EN
  • Pharmaceutical
  • Research
Detection method(s)
Due to the inclusion of the hexahistidine affinity tag, the LL-37 protein can be isolated using immobilized metal affinity chromatography. If using amylose affinity chromatography, the maltose binding protein domain may not bind efficiently and may not produce proteins with sufficient purity. Therefore, the maltose binding protein tag is not expected to contribute to protein purification (see "External links" above).
EN
Additional Information
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
Records referencing this document Show in search
Record type Field Record(s)
Country's Decision or any other Communication Living modified organism(s) 1
Living Modified Organism Related LMO(s) 2
Risk Assessment generated by a regulatory process Living modified organism(s) 1