Modification of the loops in the ligand-binding site turns avidin into a steroid-binding protein

Abstract

Background: 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.

Results: Avidin 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.

Conclusions: 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.

Figure 1

Schematic presentation of the Avd display expression constructs. (A) The phagemid constructs for Avd and Avd(N118M) display, in which Avd protein is produced solely as a fusion with pIII. (B) The phagemid constructs for Avd/Avd-pIII and Avd(N118M)/Avd(N118M)-pIII display, in which the pelB signal sequence is used for secretion of the Avd-pIII fusions and the free Avd. The cloning sites used are shown by vertical arrows.

Figure 2

Immunoblot analysis of Avd phages with anti-Avd and anti-pIII antibodies. (A) The location of the Avd-pIII fusion protein recognized by anti-Avd is indicated by the upper arrowhead. The theoretical mass of the Avd-pIII fusion protein is 38 kDa. The produced free Avd (~14 kDa) is indicated with the lower arrowhead. (B) From the immunoblot analyzed with anti-pIII antibody the full-length pIII expressed from the helper phage (VCSM13) can be seen migrating at ~60 kDa (the upper arrowhead). The Avd-pIII fusion is indicated by the middle arrowhead. In addition, some proteolytically truncated Avd-pIII forms were detected (~33 kDa; lower arrowhead). Lane 1: Avd-pIII phage; lane 2: Avd(N118M)-pIII phage; lane 3: Avd/Avd-pIII phage; lane 4: Avd(N118M)/Avd(N118M)-pIII phage. The molecular weights of standard proteins are shown as kilodaltons on the left side of each blot.

Figure 3

Three-dimensional structure of wtAvd with the loops chosen for random mutagenesis marked. One subunit of tetrameric wtAvd with bound biotin (PDB: 2AVI) [16] is shown in the figure. The randomized amino acid residues are N12, D13, L14, G15, and S16 (shown in blue) in the loop between β-strands 1 and 2, and T35, A36, V37, and T38 (shown in purple) in the loop between β-strands 3 and 4.

Figure 4

Determination of ligand-binding specificity of sbAvd-1 and sbAvd-2 proteins by microplate analysis. The binding of sbAvd-1 and sbAvd-2 to a set of different small ligands was detected using polyclonal anti-avidin antibody as a probe (A) and the effect of free biotin (10 μM) to ligand-binding was analyzed (B). Abbreviations used in the figure: PBS, Phosphate buffered saline; HSA, human serum albumin; PRO, HSA-conjugated progesterone; HYD, HSA-conjugated hydrocortisone; TES, HSA-conjugated testosterone; CHO, HSA-conjugated cholic acid; TES-B, BSA-conjugated testosterone; BTN, BSA-conjugated D-biotin; BSA, bovine serum albumin.

Figure 5

Inhibition analysis of sbAvd-1 and sbAvd-2 proteins by the SPR method. The binding of the sbAvd-1 and the sbAvd-2 to a CM5 sensor chip functionalized with testosterone-BSA was measured in the presence of 50 μM inhibitors. (A) The binding of the sbAvd-1 protein was totally inhibited by dehydroepiandrosterone, androstenedione, and biotin. (B) The binding of the sbAvd-2 protein was totally inhibited by dehydroepiandrosterone or testosterone, but not biotin. This result is due to the markedly decreased affinity of the protein towards biotin. Samples: Protein sample, green; protein with estradiol, dark blue; protein with DHT, olive; protein with testosterone, black; protein with DHEAS, blue; protein with androstenedione, brown; protein with biotin, red.

Figure 6

Analysis of sbAvd protein-testosterone interaction by MRFS method (A) A schematic representation of the experimental assembly used in the analyses. Testosterone was tethered to the AFM tip using a flexible PEG crosslinker. The steroid-binders sbAvd-1 and sbAvd-2 were covalently bound to the mica surface via a short homobifunctional spacer. (B) The force-distance cycle of the sbAvd-1 - testosterone interaction showing an unbinding event. The typical non-linear shape of the event results from the elastic properties of the PEG linker. (C) The force-distance cycle showing a sbAvd-2 - testosterone bond dissociation. Insets in (B) and (C) represent force-distance cycles in which the protein-testosterone interaction is inhibited with free testosterone.