Fusion protein capable of degrading amyloid beta peptide
A technology of fusion protein and amyloid, applied in the field of neurodegenerative diseases and medical treatment
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Embodiment 1
[0158] Description of protein domains
[0159] The extracellular domain of neprilysin is defined as amino acids 51-749 (excluding the first methionine) (SEQ ID NO: 1-4). There are two polymorphisms leading to the identified amino acid differences in this domain, and the amino acid sequences of the different variants are depicted in SEQ ID NO: 1-4.
[0160] IDE (Insulin Degrading Enzyme) is a 1018 amino acid long protein (SEQ ID NO: 5). Splice variants and polymorphic variants of IDE are described. In one splice variant, one exon was replaced with another exon of the same size, which encoded a peptide sequence similar to the "wt" exon (described in SEQ ID NO: 6). This variant has been described to be less efficient at degrading insulin and A[beta]. There are several polymorphisms in the described IDE gene which cause amino acid differences identified in this domain: D947N, E612K, L298F and E408G (numbering according to SEQ ID NO: 5). All combinations of these polymorphisms ...
Embodiment 2
[0164] Instructions for construction of the gene encoding the fusion protein Fc-neprilysin
[0165] The gene encoding the extracellular domain of neprilysin fused to the gene encoding the Fc domain of IgG2 was produced synthetically (GeneArt). The complete gene (encoding Fc-neprilysin) including the signal sequence was transferred from the GeneArt vector (pCR-Script, pGA4 or pUC-Kana) to the Gateway donor vector. The Gateway Donor Vector was used to introduce the complete gene into several expression vectors. By using the Gateway system, transfer from a donor vector to an expression vector can be performed using recombination rather than restriction enzymes. The mammalian expression vectors studied are mainly pCEP4, pEAK10, pEF5 / FRT / V5-DEST and pcDNA5 / FRT / TO (Gateway adapted). All of these are standard mammalian expression vectors based on the CMV promoter (pCEP4, pEAK10 and pcDNA5 / FRT / TO) or the EF-la promoter (pEF5 / FRT / V5-DEST). After all cloning steps the gene was sequen...
Embodiment 3
[0167] Instructions for Construction of Genes Encoding Fusion Proteins Fc-IDE and Fc-ECE1
[0168] The genes encoding the enzymes IDE and ECE1 fused to the gene encoding the Fc domain of IgG2 were produced synthetically. Complete genes (encoding Fc-IDE and Fc-ECE1 fusion proteins including signal sequences) were transferred from the initial cloning vector (pCR-Script, pGA4 or pUC-Kana) to the Gateway donor vector. The Gateway Donor Vector was used to introduce the complete gene into several expression vectors. The mammalian expression vectors studied are mainly pCEP4, pEAK10, pEF5 / FRT / V5-DEST and pcDNA5 / FRT / TO (Gateway adapted). All of these are standard mammalian expression vectors based on the CMV promoter (pCEP4, pEAK10 and pcDNA5 / FRT / TO) or the EF-la promoter (pEF5 / FRT / V5-DEST). Genes were sequenced after all cloning steps to confirm DNA efficiency.
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