Proteomics analysis of co-purifying cellular proteins associated with rAAV vectors

Abstract

Recombinant adeno-associated vectors (rAAV) are commonly purified by either chromatography or equilibrium CsCl gradient. Nevertheless, even after purification various cellular proteins often associate with rAAV vector capsids. Such co-purifying cellular proteins may raise concern about safety of gene therapy. Here we report identification and characterization of the co-purifying cellular protein in the vector preparations by using a combination of two proteomics approaches, GeLC-MS (gel electrophoresis liquid chromatography-mass spectrometry) and 2DE (two-dimensional gel electrophoresis). Most prominent bands revealed by Coomassie Blue staining were mostly similar to the AAV capsid proteins. Posttranslational modifications of capsid proteins were detected by the proteomics analysis. A total of 13 cellular proteins were identified in the rAAV vectors purified by two rounds of cesium chloride gradient centrifugation, including 9 by the GeLC-MS analysis and 4 by the 2DE analysis. Selected cellular proteins were verified by western blot. Furthermore, the cellular proteins could be consistently found associated with different AAV serotypes and carrying different transgenes. Yet, the proteins were not integral components of the viral capsis since a stringent washing procedure by column purification could remove them. These co-purified proteins in AAV vector preparations may have a role in various stages of the AAV life cycle.

Figure 1. Identification of celluar proteins co-purifed with recombinant AAV vector.

A, 1DE-SDS PAGE of recombinant AAV vector. The seven marked bands were excised and processed for MALDI-TOF analysis. B, All seven bands were identified as capsid-related proteins by the sequence homology. The Mowse score and the number of peptides matched were from the matching results against capsid protein VP1.

Figure 2. 2DE map of recombinant AAV vector.

The top 2DE map was obtained using a wide pH range of IPG strip pH–10, and the bottom one using a narrow pH range of IPG strip pH 4–7. The circled and numbered spots were identified as human proteins (Table 2), the spots only circled were identified as AAV capsid proteins.

Figure 3. Western blot analysis of co-purified proteins in 293 un-infected cell lysates and purified AAV2-dsEGFP vector particles.

10×10¹⁰ viral particles were used for the western blot.

Figure 4. Detection of protein SET in different fractions of the AAV2-dsEGFP preparation.

The vector was produced by triple plasmid transfection and purified by two rounds of cesium chloride ultra-centrifugation. The fractions with different densities were collected after the second round of ultra-centrifugation. Protein form 1×10¹⁰ viral particles was resolved on 10% SDS/PAGE. The full AAV vector particles have buoyant densities in CsCl from 1.41 to 1.45 g/cm³ while empty particles have the density of 1.32 g/cm³. A, silver staining; and B, western blot.

Figure 5. The effect of AAV serotypes, transgenes and purification methods on the presence of SET proteins in the rAAV vector preparations.

Protein from 1×1010 viral particles was resolved on 10% SDS/PAGE. AAV2-EGFP/C and AAV2-FIX/C were purified by ion exchange chromatography. A, silver staining; and B, western blot.