Abstract
BackgroundEngineered proteins, with non-immunoglobulin scaffolds, have become an important alternative to antibodies in many biotechnical and therapeutic applications. When compared to antibodies, tailored proteins may provide advantageous properties such as a smaller size or a more stable structure.ResultsAvidin is a widely used protein in biomedicine and biotechnology. To tailor the binding properties of avidin, we have designed a sequence-randomized avidin library with mutagenesis focused at the loop area of the binding site. Selection from the generated library led to the isolation of a steroid-binding avidin mutant (sbAvd-1) showing micromolar affinity towards testosterone (Kd ~ 9 μM). Furthermore, a gene library based on the sbAvd-1 gene was created by randomizing the loop area between β-strands 3 and 4. Phage display selection from this library led to the isolation of a steroid-binding protein with significantly decreased biotin binding affinity compared to sbAvd-1. Importantly, differential scanning calorimetry and analytical gel-filtration revealed that the high stability and the tetrameric structure were preserved in these engineered avidins.ConclusionsThe high stability and structural properties of avidin make it an attractive molecule for the engineering of novel receptors. This methodology may allow the use of avidin as a universal scaffold in the development of novel receptors for small molecules.
Highlights
Engineered proteins, with non-immunoglobulin scaffolds, have become an important alternative to antibodies in many biotechnical and therapeutic applications
The high stability and structural properties of avidin make it an attractive molecule for the engineering of novel receptors. This methodology may allow the use of avidin as a universal scaffold in the development of novel receptors for small molecules
In the first display construct, Avd was produced solely as a fusion with pIII (Avd-pIII; Figure 1A), whereas in the Avd/Avd-pIII display construct, free Avd subunits were produced in addition to the pIII fusion (Figure 1B)
Summary
Engineered proteins, with non-immunoglobulin scaffolds, have become an important alternative to antibodies in many biotechnical and therapeutic applications. When compared to antibodies, tailored proteins may provide advantageous properties such as a smaller size or a more stable structure. Antibodies are the most widely used biomolecules for therapeutic, diagnostic and research applications, because they can be generated against virtually any molecule using protein engineering techniques (for a review see [1]). For example the size of the antibody molecule has been reduced by producing single-domain antigen-binding derivatives [8]. Avd provides an attractive robust scaffold for the development of novel receptors, and Avd has many advantageous properties such as high chemical and thermal stability, a deep ligand binding site optimized for the binding of small molecules, and an oligomeric nature enabling signal amplification. Engineered Avd forms, in which the two pairs of the binding sites (dual-chain Avd) [19,20,21] or all four binding sites (single-chain Avd) [22] can be independently manipulated, have been developed
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