Martin Langer

Detection of rViscumin in plasma samples by immuno-PCR.

To allow for pharmacokinetic studies in adjunction with the current clinical developments of the potent cytostatic anti-cancer drug rViscumin, a sandwich immuno-PCR (IPCR) assay was developed for the detection of rViscumin in blood plasma. The IPCR was carried out with a commercially available reagent kit, consisting of pre-assembled rViscumin-specific antibody-DNA conjugates as well as a specific competitor DNA fragment to be amplified by PCR. Various combinations of capture- and detection-antibodies were compared for performance in IPCR. Using the optimized assay, as few as 50 zeptomol (approx. 100 fg/ml) rViscumin (MW 57 kDa) was detectable in standardized human serum samples. The IPCR assay was very selective for rViscumin and in spiking experiments in proband plasma samples, signal recovery rates between 70% and 120% were obtained. The linear sensitivity range of the assay covered more than five orders of magnitude. Repeated measurements of rViscumin resulted in a mean standard deviation value of 14.2%.

Investigation of charge variants of rViscumin by two‐dimensional gel electrophoresis and mass spectrometry

A method for the analysis of the rViscumin heterodimer (recombinant mistletoe lectin) based on two‐dimensional (2‐D) polyacrylamide gel electrophoresis and mass spectrometry was developed and used for quality control concerning purity and homogeneity of the recombinant protein processed under Good Manufacturing Practice (GMP) conditions. A series of spots with different pI‐values in the pH‐gradient of both rViscumin A‐ and B‐chain were observed independently from the experimental conditions like urea concentration, heat treatment or the use of cysteine alkylating agents. Comparative studies of the major spots using matrix assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS), liquid chromatography‐electrospray ionization (LC‐ESI)‐MS and LC‐ESI‐tandem MS (MS/MS) after tryptic in‐gel digestion resulted in a sequence coverage of 92% for the A‐chain and 95% for the B‐chain. No molecular differences like common chemical or post‐translational modifications or nonenzymatic deamidation were found to cause the different charge values of the separated spots. Therefore, these protein spots were extracted from the 2‐D gel and separated again by 2‐D gel electrophoresis (termed Re‐2‐DE). Each of the single spots tested in the Re‐2‐DE experiment split up in the same heterogeneous pattern concerning the pI‐values. We suggest that the observed charge variants of rViscumin are the result of conformational protein variants, existing in an equilibrium during sample preparation and/or isoelectric focusing and are not caused from microheterogeneity in the primary structure of rViscumin.

Biotechnologische Gewinnung von Seltenen Erden

Rare earth elements (REE) are ubiquitous on earth and several REE-enriched deposits are known. However, their exploitation is technically and economically challenging. Recovery and raffination processes are highly complex and generally not very sustainable. In many high tech products, however, REE constitute a crucial compound that can rarely sometimes be substituted by other elements. Novel biological approaches offer a promising option in the quest for safe, reliable, and economic REE winning processes.

Cytotoxic activity of recombinant bFGF-rViscumin fusion proteins

A fusion protein (bFGF-rMLA), containing the mitogen basic fibroblast growth factor (bFGF) and the cytotoxic component of rViscumin (recombinant mistletoe lectin), the enzymatic A-chain (rMLA), was expressed in Escherichia coli, purified, and functionally characterized. bFGF-rMLA is cytotoxic for mouse B16 melanoma cells expressing the FGF receptor with an IC(50) value of approximately 1 nM. rMLA shows no significant effect on the viability of the B16 cells up to a concentration of 141 nM. Additionally, bFGF-rMLA was associated with the rViscumin B-chain (rMLB) in an in vitro folding procedure. The IC(50) value of bFGF-rMLA/rMLB to B16 cells in the presence of lactose-to block rMLB lectin activity-was 134 pM. Thus, it was possible to enhance the efficacy of a rViscumin A-chain mitotoxin through addition of rMLB. We conclude that rViscumin fusion proteins may be generally applicable for the receptor-specific inactivation of target cells and point out their potential in drug development.