Stabilized alpha-galactosidase and uses thereof
A technology of galactosidase and galactose, applied in the field of polyprotein structure, can solve the problems of inability to start treatment, inability to provide solutions, inability to terminate, etc.
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[0395] Reference is now made to the following examples, which together with the foregoing description illustrate certain embodiments of the invention in a non-limiting manner.
[0396] Materials and methods
[0397] Material:
[0398] PEG from Iris Biotech GmbH 8 and 2000 Daltons (PEG 45 ) in PEG form, and from Pierce's PEG 5 Di-N-succinimide-poly(ethylene glycol) (di-NHS-PEG) was obtained in the form and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 25 mg / ml;
[0399] Citric acid was obtained from Sigma;
[0400]Coomassie Blue G250 from Bio-Rad;
[0401] Dimethylsulfoxide was obtained from Sigma;
[0402] D-(+)-galactose was obtained from Sigma;
[0403] Human plasma (K3 EDTA) was obtained from Bioreclamation Inc.;
[0404] 4-Methylumbelliferone was obtained from Sigma;
[0405] 4-Methylumbelliferone-A-D-galactopyranoside was obtained from Sigma;
[0406] N-Lauryl-nitrobenzoxadiazole-ceramide trihexoside (Gb 3 -NBD);
[0407] 2-(N-morphinyl)ethanesu...
Embodiment I
[0460] In Vitro Stability of Recombinant α-GAL
[0461] The in vitro stability of recombinant α-GAL was measured under various conditions as described above in the Materials and Methods section. testing of plant recombinant human α-GAL-I, and with Commercial recombinant human α-GAL.
[0462] Such as figure 1 As shown, all assay types of α-GAL showed loss of activity under simulated lysosomal conditions.
[0463] Additionally, if figure 2 As shown in , all assay types of α-GAL showed a loss of activity under simulated physiological conditions. As further shown there, under such conditions, the presence of 100 mg / ml galactose partially protected the activity of plant recombinant α-GAL-I.
[0464] Similarly, as in image 3 As shown in , in human plasma at 37°C, all tested types of α-GAL showed a loss of activity.
[0465] Such as Figure 4 It was shown that the presence of 100 mg / ml galactose partially protected the activity of plant recombinant α-GAL-I under simulate...
Embodiment II
[0469] Cross-linking of plant recombinant human α-GAL-I with di-N-hydroxysuccinimide-poly(ethylene glycol) (di-NHS-PEG) reagent
[0470] Plant recombinant human α-GAL-I (prh-α-GAL-I) was mixed with di-N-hydroxysuccinimide-poly (ethylene glycol) (di-NHS-PEG), that is, di-NHS-PEG 5 2-NHS-PEG 8 or Di-NHS-PEG 45 (di-NHS-PEG with 2000 Dalton PEG) cross-linked at Figure 5 Its structure is shown in .
[0471] Di-NHS-PEG can be attached to a protein at two sites on the protein, or at one site on the protein (eg, a lysine residue), thus forming a crosslink. exist Image 6 Both forms of connection are described in .
[0472] Add 100 μg of α-GAL-I in 28.5 μl of 2-(N-morphinyl)ethanesulfonic acid (MES) buffer (25 mM, pH 6) to 13.5 μl of phosphate buffer containing 100 mg / ml galactose (100mM, pH 8).
[0473] Di-NHS-PEG in 8 μl DMSO 5Add to α-GAL-I solution (1:50 molar ratio of 27.4 μg of α-GAL-I solution, 1:100 molar ratio of 54.8 μg of α-GAL-I solution, and 1:200 molar ratio of ...
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