Methods of inhibiting cataracts and presbyopia
A technology for cataracts and presbyopia, applied in electrotherapy, pharmaceutical formulations, medical devices, etc.
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Embodiment 1
[0074] Example 1: Cloning of γ-crystallin
[0075] [gamma]D- and [gamma]S-crystallin DNA sequences are in the pQe1 plasmid provided by the King laboratory at the Massachusetts Institute of Technology (Cambridge, MA). For purification purposes, the γ-crystallin protein sequence contains a 6×N-terminal histidine tag (His tag). This plasmid was transformed into a clonal competent cell line to create additional plasmid DNA. The plasmid was subsequently transformed into an expression competent cell line (TAM1 E. coli cells (ActiveMotif. Carlsbad, CA)) for protein synthesis.
[0076] Chemical transformation of γ-crystallin DNA into M15pRep E. coli cells for protein synthesis. Grow 1 L of culture for protein purification. The gamma-crystallin protein was purified by Ni affinity chromatography. The N-terminal His-tag contained on the γ-crystallin protein binds preferentially to the Ni column. Bound protein can be eluted using an imidazole gradient that competitively binds to Ni, ...
Embodiment 2
[0077] Example 2: Effect of pH and salt on purified γD- and γS-crystallin
[0078] Since pH and salt affect the aggregation of β-crystallin, the effect of pH and salt on γ-crystallin was also investigated. Particle size was measured using dynamic light scattering (DLS).
[0079] Experimental dynamic light scattering (DLS) was measured using an ALV goniometer with an ALV-5000 / E correlator equipped with 288 channels (ALV, Langen, Germany) and a 2W argon laser (Coherent Inc., Santa Clara ,CA), with an operating power of approximately 40mW. at angles between 30° and 90° at intervals of 5° (corresponding to 8.41×10 6 to 2.30×10 7 m -1 The scattered wave vector (q) range between) measures the scattered intensity. The scattered wave vector is defined as q=4πnsin(θ / 2) / λ, where θ is the scattering angle, and λ=514.5, the wavelength of the argon laser in vacuum, and n is 1.33, the refractive index of water. The temperature of the sample was maintained at a constant temperature of ...
Embodiment 3
[0108] Example 3 - Chemical modification of γD- and γS-crystallin with PEG24
[0109] The first modification of γ-crystallin was performed using NHSPEG24. Amino acids containing primary amines, lysine, and arginine can undergo nucleophilic substitution on N-hydroxysuccinimide (NHS)-functionalized polyethylene glycol (PEG). NHS is the active ester to promote the Sn2 reaction mechanism because it is a good leaving group. Nucleophilic substitution yields proteins modified with PEG or pegylated γ-crystallin. Pegylation was chosen because it has been shown that modifying proteins with PEG to increase solubility does not affect three-dimensional structure or properties. Specifically, PEG24 was chosen because of its reasonable molecular weight (1100.39) and spacer arm length (8.82 nm) for the added PEG24.
[0110]
[0111] NHSPEG24
[0112] Modification of γ-crystallin with PEG24 was successful, as evidenced by the large increase in the molecular weight of γ-crystallin observe...
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