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  <doc>
    <id>64794</id>
    <completedYear/>
    <publishedYear>2025</publishedYear>
    <thesisYearAccepted/>
    <language>eng</language>
    <pageFirst>1</pageFirst>
    <pageLast>22</pageLast>
    <pageNumber/>
    <edition/>
    <issue/>
    <volume/>
    <type>preprint</type>
    <publisherName>Preprints.org</publisherName>
    <publisherPlace/>
    <creatingCorporation/>
    <contributingCorporation/>
    <belongsToBibliography>0</belongsToBibliography>
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    <publishedDate>--</publishedDate>
    <thesisDateAccepted>--</thesisDateAccepted>
    <title language="eng">Corundum Particles as Trypsin Carrier for Efficient Protein Digestion</title>
    <abstract language="eng">Reusable enzyme carriers are valuable for proteomic workflows, yet many supports are expensive or lack robustness. This study describes the covalent immobilization of recombinant trypsin on micrometer-sized corundum particles and assesses their performance in protein digestion and antibody analysis. The corundum surface was cleaned with potassium hydroxide, silanized with 3-aminopropyltriethoxysilane and activated with glutaraldehyde. Recombinant trypsin was then attached, and the resulting imines were reduced with sodium cyanoborohydride. Aromatic amino acid analysis (AAAA) estimated an enzyme loading of approximately 1 µg/mg. Non-specific adsorption of human plasma proteins was suppressed by blocking residual aldehydes with a Tris-glycine-lysine buffer. Compared with free trypsin, immobilization shifted the temperature optimum from 50 to 60 °C and greatly improved stability in 1 M guanidinium hydrochloride. Activity remained above 80 % across several reuse cycles, and storage at 4 °C preserved functionality for weeks. When applied to digesting the NISTmAb, immobilized trypsin provided peptide yields and sequence coverage comparable to soluble enzyme and outperformed it at elevated temperatures. MALDI-TOF MS analysis of Herceptin digests yielded fingerprint spectra that correctly identified the antibody and achieved &gt;60 % sequence coverage. The combination of low cost, robustness and analytical performance makes corundum-immobilized trypsin an attractive option for research and routine proteomic workflows.</abstract>
    <identifier type="doi">10.20944/preprints202510.2002.v1</identifier>
    <identifier type="urn">urn:nbn:de:kobv:b43-647944</identifier>
    <enrichment key="opus.source">publish</enrichment>
    <enrichment key="opus.doi.autoCreate">false</enrichment>
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    <licence>Creative Commons - CC BY - Namensnennung 4.0 International</licence>
    <author>Sarah Döring</author>
    <author>Birte S. Wulfes</author>
    <author>Aleksandra Atanasova</author>
    <author>Carsten Jaeger</author>
    <author>Leopold Walzel</author>
    <author>Georg Tscheuschner</author>
    <author>Sabine Flemig</author>
    <author>Kornelia Gawlitza</author>
    <author>Ines Feldmann</author>
    <author>Zoltán Konthur</author>
    <author>Michael G. Weller</author>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Aluminum oxide</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Mass spectrometry</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Enzyme immobilization</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Antibodies</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Protein quantification</value>
    </subject>
    <collection role="ddc" number="543">Analytische Chemie</collection>
    <collection role="ddc" number="620">Ingenieurwissenschaften und zugeordnete Tätigkeiten</collection>
    <collection role="institutes" number="">1 Analytische Chemie; Referenzmaterialien</collection>
    <collection role="institutes" number="">1.5 Proteinanalytik</collection>
    <collection role="institutes" number="">1.8 Umweltanalytik</collection>
    <collection role="institutes" number="">1.9 Chemische und optische Sensorik</collection>
    <collection role="institutes" number="">4 Material und Umwelt</collection>
    <collection role="institutes" number="">4.2 Material-Mikrobiom Wechselwirkungen</collection>
    <collection role="themenfelder" number="">Material</collection>
    <collection role="themenfelder" number="">Chemie und Prozesstechnik</collection>
    <collection role="themenfelder" number="">Chemische Charakterisierung und Spurenanalytik</collection>
    <collection role="fulltextaccess" number="">Datei für die Öffentlichkeit verfügbar ("Open Access")</collection>
    <collection role="literaturgattung" number="">Graue Literatur</collection>
    <collection role="themenfelder" number="">Advanced Materials</collection>
    <collection role="unnumberedseries" number="">Preprints der BAM</collection>
    <thesisPublisher>Bundesanstalt für Materialforschung und -prüfung (BAM)</thesisPublisher>
    <file>https://opus4.kobv.de/opus4-bam/files/64794/preprints202510.2002.v1.pdf</file>
  </doc>
  <doc>
    <id>57567</id>
    <completedYear/>
    <publishedYear>2023</publishedYear>
    <thesisYearAccepted/>
    <language>eng</language>
    <pageFirst>1</pageFirst>
    <pageLast>18</pageLast>
    <pageNumber/>
    <edition/>
    <issue>2</issue>
    <volume>12</volume>
    <type>article</type>
    <publisherName>MDPI</publisherName>
    <publisherPlace>Basel, Schweiz</publisherPlace>
    <creatingCorporation/>
    <contributingCorporation/>
    <belongsToBibliography>1</belongsToBibliography>
    <completedDate>--</completedDate>
    <publishedDate>--</publishedDate>
    <thesisDateAccepted>--</thesisDateAccepted>
    <title language="eng">Efficient Purification of Polyhistidine-Tagged Recombinant Proteins Using Functionalized Corundum Particles</title>
    <abstract language="eng">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.</abstract>
    <abstract language="deu">Die immobilisierte Metallaffinitätschromatographie (IMAC) ist eine weit verbreitete Technik zur Reinigung von rekombinanten Proteinen mit Polyhistidin-Markierung. Sie hat jedoch oft praktische Einschränkungen, die komplexe Optimierungen und zusätzliche Schritte für die Aufreinigung erfordern. In dieser Studie stellen wir funktionalisierte Korundpartikel als neuartige, effiziente und wirtschaftliche Methode zur Reinigung rekombinanter Proteine in einem säulenfreien Format vor. Die Korundoberfläche wird mit dem Aminosilan APTES und anschließend mit EDTA-Dianhydrid modifiziert und dann mit Nickelionen beladen. Wir haben den Kaiser-Test zur Überwachung des Modifizierungsprozesses und ICP-MS zur Quantifizierung der Metallbindungskapazität verwendet. Zur Charakterisierung des Systems verwendeten wir His-markiertes Protein A/G (PAG) in Kombination mit Rinderserumalbumin (BSA). Die Korundpartikel wiesen eine Bindungskapazität von etwa 3 mg Protein pro Gramm Korund oder 2,4 mg pro 1 ml Korundsuspension auf. Als Beispiel für eine komplexe Matrix untersuchten wir auch Zytoplasma, das aus verschiedenen E. coli-Stämmen gewonnen wurde. Die Variation der Imidazolkonzentration in den Lade- und Waschpuffern zeigte, dass höhere Konzentrationen beim Laden die Reinheit verbesserten. Selbst bei einem Probenvolumen von bis zu einem Liter konnten wir rekombinante Proteine von 1 µg/mL isolieren. Beim Vergleich des Korundmaterials mit Standard-Ni-NTA-Agarose-Beads stellten wir einen höheren Reinheitsgrad mit Korund fest. Wir reinigten erfolgreich His6-MBP-mSA2 aus E. coli-Zytoplasma, ein Fusionsprotein, das aus monomerem Streptavidin und Maltose-bindendem Protein besteht. Wir reinigten auch SARS-CoV-2-S-RBD-His8, das in humanen Expi293F-Zellen exprimiert wurde, und bestätigten damit die Eignung des Materials für Zellkulturüberstände von Säugetieren. Die Materialkosten für nickelbeladenen Korund (ohne Regenerierung) werden auf weniger als 30 Cent pro Gramm funktionalisierten Trägers oder 10 Cent pro Milligramm isolierten Proteins geschätzt. Ein weiterer Vorteil dieses Systems ist die außergewöhnliche physikalische und chemische Stabilität der Korundpartikel. Insgesamt haben wir gezeigt, dass dieses neuartige Material eine effiziente, robuste und kostengünstige Reinigungsplattform für His-markierte Proteine bietet, selbst bei schwierigen, komplexen Matrices und großen Probenmengen mit niedrigen Produktkonzentrationen. Diese Methode könnte sowohl in kleinen Labors als auch in der Großindustrie eingesetzt werden.</abstract>
    <parentTitle language="eng">BioTech</parentTitle>
    <identifier type="doi">10.3390/biotech12020031</identifier>
    <identifier type="urn">urn:nbn:de:kobv:b43-575672</identifier>
    <enrichment key="opus.source">publish</enrichment>
    <enrichment key="date_peer_review">01.06.2023</enrichment>
    <enrichment key="opus.doi.autoCreate">false</enrichment>
    <enrichment key="opus.urn.autoCreate">true</enrichment>
    <licence>Creative Commons - CC BY - Namensnennung 4.0 International</licence>
    <author>Jule L. Völzke</author>
    <author>Sarah Smatty</author>
    <author>Sarah Döring</author>
    <author>Shireen Ewald</author>
    <author>Marcus Oelze</author>
    <author>Franziska Fratzke</author>
    <author>Sabine Flemig</author>
    <author>Zoltán Konthur</author>
    <author>Michael G. Weller</author>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Aluminum oxide</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Sapphire</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Ethylenediaminetetraacetic acid</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Nickel chelate</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>EDTAD</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>HexaHis-Tag</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>His6</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>6xHis</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>His8</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Bioseparation</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>IMAC purification</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Immunocapture</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Affinity chromatography</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Carrier</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Nickel</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Recombinant protein</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Escherichia coli</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Bacterial lysates</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Cytoplasm</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Polishing</value>
    </subject>
    <subject>
      <language>eng</language>
      <type>uncontrolled</type>
      <value>Downstream processing</value>
    </subject>
    <collection role="ddc" number="543">Analytische Chemie</collection>
    <collection role="ddc" number="620">Ingenieurwissenschaften und zugeordnete Tätigkeiten</collection>
    <collection role="institutes" number="">1 Analytische Chemie; Referenzmaterialien</collection>
    <collection role="institutes" number="">1.1 Anorganische Spurenanalytik</collection>
    <collection role="institutes" number="">1.5 Proteinanalytik</collection>
    <collection role="institutes" number="">1.8 Umweltanalytik</collection>
    <collection role="themenfelder" number="">Material</collection>
    <collection role="themenfelder" number="">Chemie und Prozesstechnik</collection>
    <collection role="themenfelder" number="">Chemische Charakterisierung und Spurenanalytik</collection>
    <collection role="literaturgattung" number="">Verlagsliteratur</collection>
    <collection role="fulltextaccess" number="">Datei für die Öffentlichkeit verfügbar ("Open Access")</collection>
    <collection role="unnumberedseries" number="">Wissenschaftliche Artikel der BAM</collection>
    <collection role="themenfelder" number="">Materialdesign</collection>
    <thesisPublisher>Bundesanstalt für Materialforschung und -prüfung (BAM)</thesisPublisher>
    <file>https://opus4.kobv.de/opus4-bam/files/57567/Voelzke-2023-BioTech.pdf</file>
    <file>https://opus4.kobv.de/opus4-bam/files/57567/Voelzke-2023-BioTech-Supplement.pdf</file>
  </doc>
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