Production and Purification of Adeno-Associated Viral Vectors (AAVs) Using Orbitally Shaken HEK293 Cells.

Here, we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium using 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 three-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 7-day batch process, cell cultures are further processed using a set of methods for cell lysis and vector recovery. Methods for the purification of viral particles are described, including immunoaffinity and anion-exchange chromatography, ultrafiltration, as well as digital PCR to quantify the concentration of vector particles.

Characterization of the chemokine CXCL11-heparin interaction suggests two different affinities for glycosaminoglycans.

Chemokines orchestrate the migration of leukocytes in the context of homeostasis and inflammation. In addition to interactions of chemokines with receptors on migrating cells, these processes require interactions of chemokines with glycosaminoglycans (GAGs) for cell surface localization. Most chemokines are basic proteins with Arg/Lys/His residue clusters functioning as recognition epitopes for GAGs. In this study we characterized the GAG-binding epitopes of the chemokine I-TAC/CXCL11. Four separate clusters of basic residues were mutated to alanine and tested for their ability to bind to GAGs in vitro and to activate the receptor, CXCR3. Mutation of a set of basic residues in the C-terminal helix (the 50s cluster, (57)KSKQAR(62)) along with Lys(17), significantly impaired heparin binding in vitro, identifying these residues as components of the dominant epitope. However, this GAG mutant retained nearly wild type receptor binding affinity, and its ability to induce cell migration in vitro was only mildly perturbed. Nevertheless, the mutant was unable to induce cell migration in vivo, establishing a requirement of CXCL11 for GAG binding for in vivo function. These studies also led to some interesting findings. First, CXCL11 exhibits conformational heterogeneity, as evidenced by the doubling of peaks in its HSQC spectra. Second, it exhibits more than one affinity state for both heparin and CXCR3, which may be related to its structural plasticity. Finally, although the binding affinities of chemokines for GAGs are typically weaker than interactions with receptors, the high affinity GAG binding state of CXCL11 is comparable with typical receptor binding affinities, suggesting some unique properties of this chemokine.

Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells.

Glycosylation is one of the most common posttranslational modifications of proteins. It has important roles for protein structure, stability and functions. In vivo the glycostructures influence pharmacokinetics and immunogenecity. It is well known that significant differences in glycosylation and glycostructures exist between recombinant proteins expressed in mammalian, yeast and insect cells. However, differences in protein glycosylation between different mammalian cell lines are much less well known. In order to examine differences in glycosylation in mammalian cells we have expressed 12 proteins in the two commonly used cell lines HEK and CHO. The cells were transiently transfected, and the expressed proteins were purified. To identify differences in glycosylation the proteins were analyzed on SDS-PAGE, isoelectric focusing (IEF), mass spectrometry and released glycans on capillary gel electrophoresis (CGE-LIF). For all proteins significant differences in the glycosylation were detected. The proteins migrated differently on SDS-PAGE, had different isoform patterns on IEF, showed different mass peak distributions on mass spectrometry and showed differences in the glycostructures detected in CGE. In order to verify that differences detected were attributed to glycosylation the proteins were treated with deglycosylating enzymes. Although, culture conditions induced minor changes in the glycosylation the major differences were between the two cell lines.

Purification of the extracellular domain of the membrane protein GlialCAM expressed in HEK and CHO cells and comparison of the glycosylation.

Adhesion molecules are essential for a wide range of biological and physiological functions, including cell-cell interactions, cell interactions with the extracellular matrix, cell migration, proliferation and survival. Defects in cell adhesion have been associated with pathological conditions such as neoplasia, and neurodegenerative diseases. We have identified a new adhesion molecule of the immunoglobulin family, GlialCAM. The same protein was recently published under the name hepaCAM and was suggested to be associated with hepatocellular carcinoma. Here we have expressed and purified the extracellular domain of this molecule in two mammalian expression systems, HEK and CHO cells. A three step purification protocol gave an over 95% pure protein. The extracellular domain of GlialCAM possesses several potential N- and O-glycosylation sites. Glycosylation is one of the most common post-translational modifications of secreted proteins and of the extracellular domains of membrane bound proteins. It can influence both the activity and the stability of the protein. The glycosylation pattern has been shown to depend on the cell type where the protein is expressed. We examined if differences in the glycosylation of this protein could be detected when it was expressed in the two commonly used mammalian expression systems, HEK and CHO. Differences in the glycosylation were detected.

Identification of the glycosaminoglycan binding site of the CC chemokine, MCP-1: implications for structure and function in vivo.

In a recent study, we demonstrated that glycosaminoglycan (GAG) binding and oligomerization are essential for the in vivo function of the chemokines MCP-1/CCL2, RANTES/CCL5, and MIP-1beta/CCL4 (1). Binding to the GAG chains of cell surface proteoglycans is thought to facilitate the formation of high localized concentrations of chemokines, which in turn provide directional signals for leukocyte migration. To understand the molecular details of the chemokine-GAG interaction, in the present study we identified the GAG binding epitopes of MCP-1/CCL2 by characterizing a panel of surface alanine mutants in a series of heparin-binding assays. Using sedimentation equilibrium and cross-linking methods, we also observed that addition of heparin octasaccharide induces tetramer formation of MCP-1/CCL2. Although MCP-1/CCL2 forms a dimer in solution, both a dimer and tetramer have been observed by x-ray crystallography, providing a glimpse of the putative heparin-bound state. When the GAG binding residues are mapped onto the surface of the tetramer, the pattern that emerges is a continuous ring of basic residues encircling the tetramer, creating a positively charged surface well suited for binding GAGs. The structure also suggests several possible functional roles for GAG-induced oligomerization beyond retention of chemokines at the site of production.