Exploiting Ligand-Protein Conjugates to Monitor Ligand-Receptor Interactions

We introduce three assays for analyzing ligand-receptor interactions based on the specific conjugation of ligands to SNAP-tag fusion proteins. Conjugation of ligands to different SNAP-tag fusions permits the validation of suspected interactions in cell extracts and fixed cells as well as the establishment of high-throughput assays. The different assays allow the analysis of strong and weak interactions. Conversion of ligands into SNAP-tag substrates thus provides access to a powerful toolbox for the analysis of their interactions with proteins

Scalable Production and Purification of Adeno-Associated Viral Vectors (AAV).

Here we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium in orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI) mediated transfection of a 2-plasmid system and is specified for production in milliliter to liter scales. After PEI and plasmid DNA (pDNA) complex formation the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 3-day batch process, cell cultures are further processed using different methods for lysis and recovery. Methods for the purification of viral particles are described, including iodixanol gradient purification, immunoaffinity chromatography, and ultrafiltration, as well as quantitative PCR to quantify vector titer

Coexpression of acidic fibroblast growth factor enhances specific productivity and antibody titers in transiently transfected HEK293 cells

Recombinant proteins are of great commercial and scientific interest. However, most current production methods using mammalian cells involve the time- and labor-intensive step of creating stable cell lines. Although production methods based on transient gene expression could offer a significant improvement, transient transfection is currently still limited by low titers and low specific productivity compared to stable cell lines. To overcome these bottlenecks, we have explored the use of various growth factors to enhance specific productivity and titers in the context of transient gene expression. For that purpose, several growth factors were cloned and screened for their effect on transient gene expression in HEK293E and CHO-DG44 cells. In particular, acidic fibroblast growth factor (aFGF) was able to increase specific productivity by 60% and recombinant protein titers by 80% in HEK293E cells, while FGF9 increased titers by 250% in CHO-DG44 cells

Disassembly of polyethylenimine-DNA particles in vitro: implications for polyethylenimine-mediated DNA delivery

Here a simple in vitro assay was used to investigate the disassembly of nanoparticles of polyethylenimine (PEI) and DNA. Particles were formed with various PEIs, allowed to mature for 10 min, and then exposed to different competitors (RNA, DNA, BSA or heparin) or to different conditions of pH or osmolarity. DNA release was determined by gel electrophoresis or spectroscopy. The presence of heparin or high salt yielded complete particle disassembly for all PEIs tested. The addition of RNA to particles formed with linear PEIs or branched 2 kDa PEI resulted in rapid DNA release, but RNA induced only partial disassembly of particles formed with large branched PEIs. In the presence of competitor DNA, slow disassembly was observed with particles made with linear PEIs or branched 2 kDa PEI but not for particles made with larger branched PEIs. The presence of BSA resulted in partial disassembly of PEI-DNA particles, but acidic pH did not affect particle stability. If particles were allowed to mature longer than 10 min in NaCl, subsequent heparin-mediated DNA release decreased as the incubation time and the PEI:DNA ratio increased. However, particles that matured in culture medium were disassembled by heparin independently of maturation time or PEI:DNA ratio. It was concluded that branched PEIs have a higher affinity for DNA than linear PEIs, that the intracellular disassembly of PEI-DNA particles may involve interactions between PEI and cellular RNA, and that extended maturation of PEI-DNA particles in NaCl prior to transfection may limit the intracellular release of plasmid DNA

Scalable Production and Purification of Adeno-Associated Viral Vectors (AAV).

Here we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium in orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI) mediated transfection of a 2-plasmid system and is specified for production in milliliter to liter scales. After PEI and plasmid DNA (pDNA) complex formation the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 3-day batch process, cell cultures are further processed using different methods for lysis and recovery. Methods for the purification of viral particles are described, including iodixanol gradient purification, immunoaffinity chromatography, and ultrafiltration, as well as quantitative PCR to quantify vector titer