Chimeric avidin shows stability against harsh chemical conditions—biochemical analysis and 3D structure

Abstract

Avidin and its bacterial analog streptavidin have been widely used in applications in life sciences. Recently, we described a highly thermostable engineered avidin, called chimeric avidin, which is a hybrid of avidin and avidin-related protein 4. Here, we report a protocol for pilot-scale production in E. coli and the X-ray structure of chimeric avidin. The ligand-binding properties of chimeric avidin were explored with isothermal titration calorimetry. We found chimeric avidin to be more stable against various harsh organic solvents at elevated temperatures compared to avidin and streptavidin. The properties of chimeric avidin make it a potential tool for new applications in biotechnology.

Versatile bio-ink for covalent immobilization of chimeric avidin on sol–gel substrates

“…A codon TGC encoding for C-terminal cysteine residue was added to the DNA sequence encoding for chimeric avidin equipped with ompA signal peptide from Bordetella avium [15,16] by PCR using Cys add 5 and Cys add 3 as primers (see support information). The resulting DNA amplicon was purified by agarose gel electrophoresis and isolated using the Illustra Gel purification kit (GE Healthcare).…”

“…The resulting DNA amplicon was purified by agarose gel electrophoresis and isolated using the Illustra Gel purification kit (GE Healthcare). Following subcloning to pET101/D (Invitrogen) and DNA sequencing, the protein was expressed in BL-21(AI) cells (Invitrogen) utilizing the pilot-scale fermentation protocol previously published [16]. The protein was isolated and purified by using 2-iminobiotin affinity chromatography.…”

“…To improve the stability of avidin immobilized on the polymer surfaces, a recently developed chimeric avidin could be used as an alternative to wt avidin. Genetically engineered chimeric avidin ChiAVD(I117Y) (hereinafter referred to as chimeric avidin) has improved thermostability and resistance against chemicals when compared with wt avidin or streptavidin [15,16]. The use of chimeric avidin for the immobilization enables the manufacture of sensor materials in which the stability and biotin-binding properties could be retained with excellent results over long storage periods or during demanding production stages.…”

Chimeric Avidin – NMR Structure and Dynamics of a 56 kDa Homotetrameric Thermostable Protein

“…ChiAVD(I117Y), hereafter referred to as ChiAVD, is a product of rational protein engineering [7], [8]. It is a hyperthermostable hybrid of avidin and avidin-related protein 4 (AVR4) [9]–[11] obtained by replacing a 23–residue segment in avidin with the corresponding segment found in AVR4 and additionally introducing an Ile to Tyr, π–π,1–3 interface–stabilizing point mutation.…”

“…Since ChiAVD has successfully been applied in novel approaches in biotechnology [12], [13], the understanding of the molecular properties of the protein is of our special interest. The structure of ChiAVD in the biotin–free form has recently been solved by X–ray crystallography [8]. In this study we present the solution NMR structure of ChiAVD in the biotin–bound form determined at 58°C.…”