Disulfide bond stabilized polypeptide compositions and methods of use
A stable and genomic technology, applied in biochemical equipment and methods, drug combination, expression enhancement stability/folded protein fusion, etc., can solve problems such as poor stability and limited half-life of therapeutic enzymes
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example 1
[0164] Example 1: Identification of amino acid residues for cysteine substitution of wild-type α-GAL
[0165] Examination of potential sites for substitution of cysteine residues in the crystal structure of dimerized α-GAL (PDB ID 3HG3), resulting in additional disulfide bonds for enhanced stability ( Figure 1A ). NAMD with CHARMM force field was used for analysis. Based on the analysis, the cysteine mutants shown in Table 8 were prepared using standard methods of site-directed mutagenesis.
[0166]
[0167] see also Figure 1B . Amino acid sequences are provided in Table 1.
example 2
[0168] Example 2: Dimerization and enzymatic activity of modified α-GAL
[0169] The formation of disulfide-bonded dimers of modified α-GAL was examined in cell lysates and media ( Figure 2A ). Clones of each α-GAL construct were transiently expressed in 293HEK cells. Cell lysates and media were run on a 4%-12% gradient SDS-PAGE and transferred to nitrocellulose. α-GAL was detected by western blotting with rabbit monoclonal anti-α-GAL 1:2000 (abcam ab168341).
[0170] Electrophoresis and Western blotting were performed on reduced and non-reduced samples. As shown in Figure 2, the M51C-G360C and D233C-I359C versions of α-GAL readily formed disulfide-bonded α-GAL dimers.
[0171] To prepare samples, 1 x 10^6 cells with transient expression of the α-GAL construct were harvested. Cells were lysed in 500 ul of 20 mM sodium phosphate buffer pH 6.5, 0.25% TX-100. Cell lysates were centrifuged at 10,000 g for 2 min, and the supernatant was transferred to a new tube. Transfer...
example 3
[0173] Example 3: Stability analysis of modified α-GAL over time in an acidic environment
[0174] To test pH stability over 24 h, transiently expressed mutant and wild-type α-GAL were captured using concanavalin A (ConA) agarose pull-down according to standard methods. Dilute the ConA eluate in pH 4.6 buffer or pH 7.4 buffer. Samples were pre-incubated at pH 4.6 or 7.4 for 0, 0.5, 1, 2, 4, 5 and 24 hours.
[0175] To measure enzyme activity, pH 4.6 buffer was added to each sample and activity was tested on 4-MUG substrate. The reaction mixture was incubated at 37°C for 1 hour. The reaction was stopped by adding 125 uL of stop buffer (0.4M Glycine-NaOH, pH 10.8) Fluorescence was read with a Spectramax plate reader: Excitation: 360nm, Emission: 450nm. The results are shown in Figure 3A middle.
[0176] For long-term stability testing, transiently expressed modified and wild-type α-GAL were isolated from culture medium, enriched and purified using ConA Sepharose beads as...
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