Expression, purification, and MALDI analysis of RPE65.
OBJECTIVE
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.
Ma
J
J
Department of Ophthalmology, Medical University of South Carolina, Charleston 29425, USA. majx@musc.edu
Zhang
J
J
Othersen
K L
KL
Moiseyev
G
G
Ablonczy
Z
Z
Redmond
T M
TM
Chen
Y
Y
Crouch
R K
RK
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002352:Carrier Proteins; D002417:Cattle; D002846:Chromatography, Affinity; D017931:DNA Primers / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005136:Eye Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D008970:Molecular Weight; D010857:Pigment Epithelium of Eye / Q000737:chemistry / Q000378:metabolism*; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000096:biosynthesis; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
D002352:Carrier Proteins; D017931:DNA Primers; D005136:Eye Proteins; D011506:Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase