Nanobody AAVX: Pan-serotype platform for AAV bioprocessing

bioRxiv

Overview

Researchers developed a pan-serotype AAV-binding nanobody, AAVX, produced in E. coli to streamline AAV purification, quantification, and detection. The platform enables high-purity protein production and versatile applications across various AAV serotypes, offering a customizable toolset for rAAV research.

In Depth

Key Findings

The development of nanobody AAVX provides a versatile, pan-serotype platform for the recombinant adeno-associated virus (rAAV) workflow. By expressing AAVX fusion constructs in E. coli, the researchers achieved high-efficiency soluble production, with a one-step immobilized metal-ion affinity chromatography (IMAC) process yielding proteins with purity levels mostly exceeding 90%.

Technical Details

The platform utilizes AAVX in several distinct configurations to address different bioprocessing needs:

• Purification: AAVX crosslinked with an agarose matrix allows for the efficient purification of intact AAV2 and AAV5 capsids from cell lysates, verified via ELISA and qPCR.
• Quantification: AAVX serves as a capture reagent in ELISA. Additionally, the team developed a rapid, label-free quantification assay for AAV capsids using tagged AAVX and streptavidin biosensors via biolayer interferometry (BLI).
• Detection: When fused to luciferase, AAVX acts as a detection reagent. For cellular visualization, an AAVX-eGFP fusion was used to image AAV capsids in fixed HEK-293 cells using fluorescence microscopy.

Background & Context

The research and production of rAAV require tools that are both rapid and cost-effective. The AAVX nanobody was designed as a fusion partner that can be cytosolically expressed in E. coli starting from an amino acid sequence, allowing for the addition of various tags to suit specific downstream applications.

Significance & Outlook

The ability to produce AAVX-based fusion proteins in E. coli ensures a readily customizable and scalable supply of tools for AAV research. By integrating purification, quantification, and detection into a single nanobody-based platform, this approach simplifies the technical requirements for handling multiple AAV serotypes in bioprocessing environments.

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