Chemische Charakterisierung und Spurenanalytik
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- nein (1)
Therapeutic monoclonal antibodies are the fastest-growing group of biological agents which generated a yearly turnover of USD 210 billion in 2022 and whose sales are expected to grow by 10% annually over the next 10 years. With steadily increasing market importance, analytical methods for reliable quantification of therapeutic antibodies also become more and more relevant. Liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) has become the main technology for antibody quantification. This approach, however, requires enzymatic digestion of the intact protein into peptides, for which a wide range of different protocols exists that often lead to different results depending on the digestion procedure or trypsin variants used. In particular, the amount and type of detergents added for protein unfolding prior to digestion is known to create significant bias in measurement results. The overall goal of the presented project is the application of novel thermostable and surface-functionalized trypsin particles for improved antibody digestion. Specifically, a trypsin-variant described in the literature exhibiting increased activity and thermal stability above 80°C, will be examined. The application of this enzyme should allow to perform digestion at elevated temperatures where the protein is naturally unfolding thereby increasing enzyme accessibility without the need for detergents. Furthermore, we will immobilize the thermostable trypsin onto the surface to further enhance enzyme stability, prevent self-digestion, and enable separation of trypsin from target peptides before LC–MS/MS analysis. As an immobilization platform, cheap and non-porous corundum particles will be used as these show high chemical stability and low levels of interaction of matrix proteins with the functionalized surface. adsorption. In a multidisciplinary collaboration with the SALSA Photonics Lab, we will investigate the characteristics of covalent enzyme binding and unspecific peptide binding using an interface-sensitive analytical tool, vibrational sum-frequency generation (VSFG) spectroscopy. The insights gained will not only lead to new competencies in peptide and enzyme surface analysis using VSFG spectroscopy in SALSA but will also significantly contribute to optimizing antibody quantification.
Immobilized metal affinity chromatography (IMAC) is a widely used technique for purifying polyhistidine-tagged recombinant proteins. However, it often has practical limitations that require complex optimizations and additional steps for purification. In this study, we introduce functionalized corundum particles as a novel, efficient, and economical method for purifying recombinant proteins in a column-free format. The corundum surface is modified with amino silane APTES, followed by EDTA dianhydride, and then loaded with nickel ions. We used the Kaiser test to monitor the modification process and ICP-MS to quantify the metal-binding capacity. To evaluate the system, we used His-tagged protein A/G (PAG) mixed with bovine serum albumin (BSA). The corundum particles exhibited a binding capacity of approximately 3 mg of protein per gram of corundum or 2.4 mg per 1 mL of corundum suspension. We also examined cytoplasm obtained from different E. coli strains as an example of a complex matrix. Varying the imidazole concentration in the loading and washing buffers showed that higher concentrations during loading improved purity. Even with sample volumes as large as one liter, we successfully isolated recombinant proteins down to a concentration of 1 µg/mL. We found higher purity levels with corundum when comparing the corundum material to standard Ni–NTA agarose beads. We successfully purified His6-MBP-mSA2, a fusion protein comprising monomeric streptavidin and maltose-binding protein, from E. coli cytoplasm, demonstrating the method's applicability. We also purified SARS-CoV-2-S-RBD-His8 expressed in human Expi293F cells, confirming its suitability for mammalian cell culture supernatants. The material cost of the nickel-loaded corundum material (without regeneration) is estimated to be less than 30 cents per gram of functionalized support or 10 cents per milligram of isolated protein. Another advantage of this system is the exceptional physical and chemical stability of corundum particles. Overall, we have demonstrated that this novel material offers an efficient, robust, and cost-effective purification platform for His-tagged proteins, even in challenging, complex matrices and large sample volumes with low product concentrations. This method has potential applications in both small laboratories and large-scale industrial settings.
Vergleichende Darstellung SARS-CoV-2-spezifischer Nanobodys aus unterschiedlichen Wirtsorganismen
(2021)
Aufgrund der anhaltenden COVID-19-Pandemie werden neutralisierende Therapeutika benötigt. Eine Möglichkeit zur Behandlung stellt die Verwendung monoklonaler Anti-SARS-CoV-2-Immun-globuline dar. Ihre Produktion in Säugetierzellen ist jedoch schwer skalierbar, um den weltweiten Bedarf zu decken. VHH-Antikörper, auch Nanobodys genannt, bieten hierfür eine Alternative, da sie eine hohe Temperaturstabilität aufweisen und eine kostengünstige Produktion in prokaryotischen Wirtsorganismen ermöglichen.