Rapid mRNA‐Display Selection of an IL‐6 Inhibitor Using Continuous‐Flow Magnetic Separation

Abstract

Wenige Auserwählte: Mithilfe von Mikrofluidiktechniken sind rasch Affinitätsreagentien zugänglich, die in einem mRNA‐Display an Zielproteine binden. Ein kontinuierliches Flussverfahren mit magnetischer Trennung (CFMS; Schritte a–h) ohne Spezialapparatur bietet reproduzierbar stringente Selektionsbedingungen. Die Methode übertraf für das Zielprotein Interleukin‐6 die Partitionseffizienz konventioneller Verfahren um einen Faktor von etwa 30.

Rapid Identification of Neutralizing Antibodies against SARS-CoV-2 Variants by mRNA Display

“…The mRNA- puromycin template was translated (Purexpress, NEB) followed by incubation with KCl (550 mM final) and MgCl2 (60 mM final) for 1 hour at room temperature to enhance fusion formation (Liu et al, 2000). The mRNA-scFv fusions were then affinity-purified using M2 anti-Flag beads (Sigma-Aldrich) to remove non-fused template and sequences containing nonsense mutations (Liao et al, 2009; Olson et al, 2011). After elution with 3XFlag peptide (Sigma-Aldrich), the fusions were reverse transcribed with super script II (ThermoFisher).…”

RasIns: Genetically Encoded Intrabodies of Activated Ras Proteins

“…The BC loop is shown in blue, while the FG loop is shown in red. Backbone mutations relative to wild-type 10FnIII are shown in yellow [15,24]. In order to direct the library to recognize active Ras, we constructed variants of e10FnIII where the BC loop was a doped sequence from a previous Ras ligand (iDab#6) [8].…”

“…The 10FnIII scaffold is small (10 kDa), lacks disulfides, can be expressed in Escherichia coli, and is an alternative to antibodies for generating affinity reagents and intrabodies [16][17][18][19][20][21][22][23]. In addition, we evolved a version of 10FnIII, termed e10FnIII, which improves solubility and expression in E. coli and in the reticulocyte lysate translation system [15,24].…”