Fusion proteins for delivery of gdnf and bdnf to the central nervous system
A compound, A-X-B technology, applied in neuregulin, nervous system diseases, DNA/RNA fragments, etc., can solve cumbersome and difficult problems
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Embodiment 1
[0156] Angiopep-2 / GDNF construct
[0157] Generate sequences including Angiopep-2 and hGDNF (hGDNF 78-211 ) construct. These constructs include the N-terminus (His) 6 Tag, thrombin cleavage site, Angiopep-2 sequence and GDNF sequence. A control peptide was also generated without the Angiopep-2 sequence ( figure 2 ). The amino acid sequences of the N-terminal portions of these sequences are shown in image 3 . The strategy used to clone these constructs is described in Figure 4-7 middle. Similar strategies can be used to generate BDNF constructs. The sequences of these constructs are shown in Figure 8-12 middle. A schematic showing the Angiopep-2-GDNF compound bound to GFRα1 is shown in Figure 13 middle.
[0158] Generate additional GDNF constructs such as Figure 14 shown in. These include hGDNF in which Angiopep-2 is attached to its N-terminus 78-211 (An2-hGDNF); hGDNF in which Angiopep-2 is attached to its C-terminus 78-211 (hGDN F-An2); hGDNF with reverse...
Embodiment 2
[0160] Receptor binding of GDNF construct conjugates
[0161] To detect receptor binding of GDNF conjugates, a double-sandwich Elisa was used. Briefly, mouse anti-human IgG antibodies were bound to one plate ( Figure 15 ). A GFRα1 receptor / IgG Fc fusion was added, which binds to these antibodies. To detect ligand binding, GDNF, GDNF conjugates, or Angiopep-2 were each added to the plates. The plates were then treated sequentially with goat anti-GDNF antibody and alkaline phosphatase-conjugated rabbit anti-goat IgG antibody. These samples were then treated with p-nitrophenyl phosphate (p-NPP), an alkaline phosphatase (AP) substrate that changes from colorless to yellow after AP treatment. Binding of proteins is detected based on this color change.
[0162] In this assay, all fusion proteins tested were able to bind the GDNF receptor at levels similar to GDNF itself ( Figure 16 ). Angiopep-2 (a negative control) was not observed to bind to this receptor. Individual bindi...
Embodiment 3
[0165] homodimer formation
[0166] As mentioned above, GDNF is known to form homodimers via disulfide bonds ( Figure 17 ). Such dimer formation was also observed with the An2-GDNF protein ( Figure 18 ). These disulfide bonds can be reduced by treatment with reducing agents such as dithiothreitol (DTT).
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