Design and Construction of Highly Stable, Protease-resistant Chimeric Avidins

Abstract

The chicken avidin gene family consists of avidin and seven separate avidin-related genes (AVRs) 1-7. Avidin protein is a widely used biochemical tool, whereas the other family members have only recently been produced as recombinant proteins and characterized. In our previous study, AVR4 was found to be the most stable biotin binding protein thus far characterized (T m ‫؍‬ 106.4°C). In this study, we studied further the biotin-binding properties of AVR4. A decrease in the energy barrier between the biotin-bound and unbound state of AVR4 was observed when compared with that of avidin. The high resolution structure of AVR4 facilitated comparison of the structural details of avidin and AVR4. In the present study, we used the information obtained from these comparative studies to transfer the stability and functional properties of AVR4 to avidin. A chimeric avidin protein, ChiAVD, containing a 21-amino acid segment of AVR4 was found to be significantly more stable (T m ‫؍‬ 96.5°C) than native avidin (T m ‫؍‬ 83.5°C), and its biotin-binding properties resembled those of AVR4. Optimization of a crucial subunit interface of avidin by an AVR4-inspired point mutation, I117Y, significantly increased the thermostability of the avidin mutant (T m ‫؍‬ 97.5°C) without compromising its high biotin-binding properties. By combining these two modifications, a hyperthermostable ChiAVD(I117Y) was constructed (T m ‫؍‬ 111.1°C). This study provides an example of rational protein engineering in which another member of the protein family has been utilized as a source in the optimization of selected properties.

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

“…Based on both sequence analysis and structural comparison the potential role of Tyr115 in AVR4 (equivalent to position 117 in avidin) in stabilizing the tetrameric structure was discussed previously ( Fig. 1; Hytönen et al, 2004bHytönen et al, , 2005. We have also found that I117Y stabilizes avidin .…”

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).…”

“…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.…”

Genetically engineered avidins and streptavidins

“…Secondly, they can serve as a source of ideas when developing advanced (strept)avidin forms. The development of a non-glycosylated, acidic avidin mutant [104] and avidin-AVR chimeras [129] are examples of the latter. As described in previous sections, modification of (strept)avidin has until recently been limited to the homotetrameric approach.…”