Purification of Functional Gene Transfer Agents Using Two-Step Preparative Monolithic Chromatography

Abstract

Background: Gene transfer agents (GTAs) are phage-like particles that transfer cellular genomic DNA between cells. A hurdle faced in studying GTA function and interactions with cells is the difficulty in obtaining pure and functional GTAs from cultures.

Materials and methods: We used a novel two-step method for purification of GTAs from R. capsulatus by monolithic chromatography.

Results: Our efficient and simple process had advantages compared to previous approaches. The purified GTAs retained gene transfer activity and the packaged DNA could be used for further studies.

Conclusions: This method is applicable to GTAs produced by other species and small phages, and could be useful for therapeutic applications.

Keywords: Convective Interaction Media®; Rhodobacter capsulatus; gene transfer; monolithic separations; nanoparticle tracking analysis; phage purification.

FIG. 1.

(A) Western blot measuring RcGTA major capsid protein (∼30 kDa). Supernatants of photoheterotrophic anaerobic cultures in RCV were harvested at stationary phase to compare GTA release. (B) SDS-PAGE (12%) comparing GTA release by DE442 in various culture media. Media were compared by the intensity of the RcGTA major capsid protein band at ∼30 kDa. (C) Representative growth curve of DE442 grown under photoheterotrophic anaerobic conditions in RCVm+MOPS at 35°C. GTA, gene transfer agent; MOPS, 3-(N-morpholino)propanesulfonic acid; RcGTA, GTA produced by R. capsulatus; RCV, Rhodobacter capsulatus V; SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

FIG. 2.

CIMmultus OH purification of DE442 GTA from culture filtrate. (A) Purification chromatogram monitoring 280 nm (primary y-axis). Number labels indicate position of collected fractions. Dashed line indicates decreasing potassium phosphate gradient (secondary y-axis). (B) SDS-PAGE (12%) of collected fractions (number labels 1–4 in A), a fraction collected from the regeneration wash (NaOH wash) and filtrate SM. OH, hydroxyl functionalized monolith; SM, starting material.

FIG. 3.

CIMmultus OH purification of DE442 GTA from culture filtrate. (A) Visible light spectra of filtrate SM and fractions corresponding to those in Fig 2A. Vertical lines at 802 and 855 nm indicate LH2 absorption peaks, indicative of presence of chromatophores. (B) Comparison of the concentration and size of measured particles in Fraction 4 through nanoparticle tracking analysis, n = 3. (C) Particle size distribution scatter of Fraction 4 through nanoparticle tracking analysis, n = 3 as indicated in legend.

FIG. 4.

CIMmultus QA purification of DE442 GTA from OH purification fraction 4. (A) Purification chromatogram monitoring 280 nm (primary y-axis). The GTA fraction was collected as indicated. The dashed line indicates increasing ammonium sulfate gradient (secondary y-axis). (B) SDS-PAGE (12%) of the collected GTA. QA, quaternary ammonium functionalized monolith.

FIG. 5.

CIMmultus QA purification of DE442 GTA from OH purification fraction 4. (A) Visible light spectra of filtrate SM, OH purification fraction 4 (OH purified), and the collected GTA peak from QA purification (QA GTA). Vertical lines at 802 and 855 nm indicate LH2 absorption peaks, indicative of presence of chromatophores. (B) Comparing the concentration and size of measured particles in collected GTAs through nanoparticle tracking analysis, n = 3. (C) Particle size distribution scatter of collected GTAs through nanoparticle tracking analysis, n = 3 as indicated in legend.

FIG. 6.

Analysis of DNA extracted from purified DE442 GTAs. (A) Agarose gel (1%) of DNA extracted from purified GTAs from two strains of Rhodobacter capsulatus. The DE442 DNA band is at ∼4 kb and the negative control DE442Δclpx lacks a DNA band. (B) Oxford Nanopore MinION read distribution histogram of DE442 GTA DNA, comparing base-called length and read density.