Overview
In the AGE-Thwart project, we aim to develop the parts for producing sRAGE protein which have the ability to bind to collagen patches . To make sRAGE bind to collagen, we design a recombinant protein which has sRAGE and Collagen binding domain (CBD) on each terminal of protein. To combine sRAGE and CBD into a recombinant protein, we separately join Npu-DnaE intein in the gene block of sRAGE and CBD, then combine them by intein splicing. Together, we believed that the intein system would contribute to the iGEM teams in the future.
Basic parts
| Part number | Name | Description | Length |
| BBa_K1222999 | Lac operator/pSB1C3 | The LacO operator is a DNA sequence that is recognized by the LacI repressor. | 25 bp |
| BBa_K3778012 | T7 promoter | T7 RNA polymerase would recognize DNA sequence of this region and start to transcript. | 19 bp |
| BBa_K3778013 | RBS (Ribosome-binding site) | Ribosome would recognize the RNA sequence of this region and start to translate. | 23 bp |
| BBa_K3782002 | 6XHis-Tag | His tag for purification and identification of protein of interest. | 18 bp |
| BBa_25RE40J5 | 6xHis (for 6xHis-(Npu-DnaEC)-CWE-CBD) | A histidine tag can be captured by Ni2+ resin, which is used for protein purification through affinity chromatography. | 18 bp |
| BBa_25KWF4OM | EGFP | EGFP is a modified form of GFP with higher fluorescence intensity and improved stability. It is commonly used as a biomarker. | 714 bp |
| BBa_K1362456 | Thrombin recognition/cleavage site | This part codes for the amino acid sequence LVPRGS. This site is recognized by Thrombin, which will cleave the Arg-Gly peptide bond. | 18 bp |
| BBa_K3778018 | T7 terminator | T7 RNA polymerase would recognize the DNA sequence of this region and stop the transcription. | 48 bp |
| BBa_25WIU0DS | Npu-DnaEC (IntC) (Codon optimized for E. Coli expression) | Intein can auto-catalytically excises itself without energy from precursor protein, and ligate two exteins with a peptide bond. | 108 bp |
| BBa_25JPVVSQ | Npu-DnaEN (IntN) (Codon optimized for E. Coli expression) | Intein can auto-catalytically excises itself without energy from precursor protein, and ligate two exteins with a peptide bond. | 305 bp |
| BBa_25T4TYKW | Npu-DnaEN (IntN) (Codon optimized for expression in human) | Intein can auto-catalytically excises itself without energy from precursor protein, and ligate two exteins with a peptide bond. | 305 bp |
| BBa_K1362433 | CWE (extein) | Cys-Trp-Gly peptide in C-terminal extein can help Npu-DnaE intein to achieve highly efficient and rapid splicing. | 9 bp |
| BBa_25L21KZ9 | RGK | Arg-Gly-Lys peptide in N-terminal extein can help Npu-DnaE intein to achieve highly efficient and rapid splicing. | 9 bp |
| BBa_25ZROENN | CBD (Lumican LRR5-7) (Codon optimized for E. Coli expression) | Lumican LRR5-7 derived from the central part of the protein has high affinity to collagen (KD ∼ 45 nm). | 204 bp |
| BBa_25LO3UHE | sRAGE (Codon optimized for E. Coli Expression) | sRAGE is soluble form of RAGE，it can serve as decoy receptor to bind with AGE and other RAGE ligands, preventing them interacting with RAGE. | 905 bp |
| BBa_25LVY9AB | sRAGE (Codon optimized for expression in human; secreted form) | sRAGE is soluble form of RAGE，it can serve as decoy receptor to bind with AGE and other RAGE ligands, preventing them interacting with RAGE. | 969 bp |
| BBa_25Q2IIB1 | mut-sRAGE (Condon optimized for expression in human; secreted form) | Research has found that mutant RAGE which has de-N-glycosylation at N25 and N81 can enhance AGE binding. | 903 bp |
| BBa_25DYJ8D8 | Linker (GGGSGGEF) | GGGSGGEF Linker can minimize steric hindrance arising from protein-protein interaction by separating two protein domains. | 24 bp |
Composite parts
| Part number | Name | Description | Length |
| BBa_250NWJIC | IntC-CWE-6xHis-CBD (Codon optimized for E. Coli expression) |
|
546 bp |
| BBa_25KRTWWB | 6xHis-IntC-CWE-CBD (Codon optimized for E. Coli expression) |
|
544 bp |
| BBa_25D3GIPS | 6xHis-Thrombin site-EGFP-linker-CBD (Codon optimized for E. Coli expression) |
|
1089 bp |
| BBa_253SWTVB | sRAGE-EGFP-6xHis-RGK-IntN (Codon optimized for E. Coli expression) |
|
2152 bp |
| BBa_25QKP8VT | Secreted sRAGE-EGFP-6xHis-RGK-IntN (Codon optimized for expression in human) |
|
2016 bp |
| BBa_25T0WI1Z | Secreted mut-sRAGE-EGFP-6xHis-RGK-IntN (Codon optimized for expression in human) |
|
1950 bp |
| BBa_25HG0XYO | IntC-6xHis-CBD (Codon optimized for expression in human) |
|
339 bp |
| BBa_25B8VLQC | 6xHis-IntC-CBD (Codon optimized for expression in human) |
|
339 bp |
| BBa_257IITHC | EGFP-6xHis-linker-CBD (Codon optimized for expression in human) |
|
960 bp |
Reference
- Osawa, M., Yamamoto, Y., Munesue, S., Murakami, N., Sakurai, S., Watanabe, T., ... & Yamamoto, H. (2007). De-N-glycosylation or G82S mutation of RAGE sensitizes its interaction with advanced glycation endproducts. Biochimica et Biophysica Acta (BBA)-General Subjects, 1770(10), 1468-1474.
- Kalamajski, S., & Oldberg, Å. (2009). Homologous sequence in lumican and fibromodulin leucine-rich repeat 5-7 competes for collagen binding. Journal of Biological Chemistry, 284(1), 534-539.
- Cheriyan, M., Pedamallu, C. S., Tori, K., & Perler, F. (2013). Faster protein splicing with the Nostoc punctiforme DnaE intein using non-native extein residues. Journal of Biological Chemistry, 288(9), 6202-6211.
- Zettler, J., Schütz, V., & Mootz, H. D. (2009). The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. FEBS letters, 583(5), 909-914.
- Oliveira, M. I. A., Souza, E. M. D., Pedrosa, F. D. O., Réa, R. R., Alves, A. D. S. C., Picheth, G., & Rego, F. G. D. M. (2013). RAGE receptor and its soluble isoforms in diabetes mellitus complications. Jornal Brasileiro de Patologia e Medicina Laboratorial, 49, 97-108.