PURPOSE: RPE65 is preferentially expressed in the retinal pigment epithelium (RPE) and is essential for retinal function. The purpose of the study was to develop methods for the expression of the protein, determine the accurate molecular weight of this expressed protein, and quantitate the amount of RPE65 in the bovine RPE. METHODS: Human RPE65 was expressed in Sf9 cells using the baculovirus system. The subcellular localization was determined by Western blot analysis and immunocytochemistry. An ELISA was developed for RPE65 and used to measure levels in bovine RPE. Recombinant and native RPE65 were purified by affinity chromatography. Molecular mass was determined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. RESULTS: Recombinant human (rH)RPE65 was expressed as a major protein associated with cell membrane in Sf9 cells. The recombinant protein was purified to apparent homogeneity from both the membrane and nonmembrane fractions. The identity of the purified protein was confirmed by Western blot analysis and by partial peptide sequencing. rHRPE65 from the nonmembrane fraction has a mass of 64,867 +/- 80 which is close to the calculated molecular weight from the amino acid sequence including the His-tag (64,663), whereas the membrane-associated rHRPE65 has a molecular mass of 65,380 +/- 150, which is significantly higher than that of the non-membrane-associated form and the calculated molecular weight, suggesting posttranslational modifications. Similarly, native RPE65 was detected in the cytosolic and microsomal fractions of the bovine RPE, with an average level of 3.8 +/- 1.3 and 7.2 +/- 0.4 microg RPE65 per eye, respectively. The cytosolic form had a molecular mass of 61,161 +/- 60, which is close to the calculated value (60,944), whereas that of the microsomal form was 61,961 +/- 170. CONCLUSIONS: RPE65 is expressed in two forms, one of which is membrane associated and contains significant posttranslational modifications, similar to the native membrane-associated form.
PURPOSE:RPE65 is preferentially expressed in the retinal pigment epithelium (RPE) and is essential for retinal function. The purpose of the study was to develop methods for the expression of the protein, determine the accurate molecular weight of this expressed protein, and quantitate the amount of RPE65 in the bovine RPE. METHODS:HumanRPE65 was expressed in Sf9 cells using the baculovirus system. The subcellular localization was determined by Western blot analysis and immunocytochemistry. An ELISA was developed for RPE65 and used to measure levels in bovine RPE. Recombinant and native RPE65 were purified by affinity chromatography. Molecular mass was determined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. RESULTS: Recombinant human (rH)RPE65 was expressed as a major protein associated with cell membrane in Sf9 cells. The recombinant protein was purified to apparent homogeneity from both the membrane and nonmembrane fractions. The identity of the purified protein was confirmed by Western blot analysis and by partial peptide sequencing. rHRPE65 from the nonmembrane fraction has a mass of 64,867 +/- 80 which is close to the calculated molecular weight from the amino acid sequence including the His-tag (64,663), whereas the membrane-associated rHRPE65 has a molecular mass of 65,380 +/- 150, which is significantly higher than that of the non-membrane-associated form and the calculated molecular weight, suggesting posttranslational modifications. Similarly, native RPE65 was detected in the cytosolic and microsomal fractions of the bovine RPE, with an average level of 3.8 +/- 1.3 and 7.2 +/- 0.4 microg RPE65 per eye, respectively. The cytosolic form had a molecular mass of 61,161 +/- 60, which is close to the calculated value (60,944), whereas that of the microsomal form was 61,961 +/- 170. CONCLUSIONS:RPE65 is expressed in two forms, one of which is membrane associated and contains significant posttranslational modifications, similar to the native membrane-associated form.
Authors: Alexander G Marneros; Douglas R Keene; Uwe Hansen; Naomi Fukai; Karen Moulton; Patrice L Goletz; Gennadiy Moiseyev; Basil S Pawlyk; Willi Halfter; Sucai Dong; Masao Shibata; Tiansen Li; Rosalie K Crouch; Peter Bruckner; Bjorn R Olsen Journal: EMBO J Date: 2003-12-11 Impact factor: 11.598
Authors: Gennadiy Moiseyev; Ying Chen; Yusuke Takahashi; Bill X Wu; Jian-Xing Ma Journal: Proc Natl Acad Sci U S A Date: 2005-08-22 Impact factor: 11.205
Authors: Marcin Golczak; Philip D Kiser; David T Lodowski; Akiko Maeda; Krzysztof Palczewski Journal: J Biol Chem Date: 2010-01-25 Impact factor: 5.157
Authors: Philip D Kiser; Marcin Golczak; David T Lodowski; Mark R Chance; Krzysztof Palczewski Journal: Proc Natl Acad Sci U S A Date: 2009-10-05 Impact factor: 11.205