Related Experiment Video
Updated: Aug 11, 2026
10:52
Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions
Published on: September 28, 2017
Expression, purification, and MALDI analysis of RPE65
1Department of Ophthalmology, Medical University of South Carolina, Charleston 29425, USA. majx@musc.edu
Investigative Ophthalmology & Visual Science
|May 31, 2001
Summary
Retinal pigment epithelium protein 65 (RPE65) is expressed in two forms, one membrane-associated with posttranslational modifications. This study developed methods to express and quantify RPE65 in bovine retinal pigment epithelium.
Area of Science:
- Biochemistry
- Molecular Biology
- Ophthalmology
Background:
- Retinal pigment epithelium protein 65 (RPE65) is crucial for retinal function.
- RPE65 is predominantly found in the retinal pigment epithelium (RPE).
Purpose of the Study:
- Develop expression methods for RPE65 protein.
- Determine the precise molecular weight of expressed RPE65.
- Quantify RPE65 levels in bovine RPE.
Main Methods:
- Expressed human RPE65 using a baculovirus system in Sf9 cells.
- Utilized Western blot, immunocytochemistry, and ELISA for analysis.
- Determined molecular mass via MALDI mass spectrometry and purified proteins using affinity chromatography.
Main Results:
- Recombinant human RPE65 was expressed and purified, with membrane-associated forms showing higher molecular weight, indicating posttranslational modifications.
- Native RPE65 in bovine RPE also presented in cytosolic and microsomal forms with distinct molecular masses.
- Quantified RPE65 levels in bovine RPE, averaging 3.8 microg/eye (cytosolic) and 7.2 microg/eye (microsomal).
Conclusions:
- RPE65 exists in at least two distinct forms.
- One form is membrane-associated and undergoes significant posttranslational modifications.
- These findings are consistent with the native membrane-associated form of RPE65.
