A novel, stable, helical scaffold as an alternative binder - construction of phage display libraries

Specific, high affinity binding macromolecules are of great importance for biomedical and biotechnological applications. The most popular classical antibody-based molecules have recently been challenged by alternative scaffolds with desirable biophysical properties. Phage display technology applied to such scaffolds allows generation of potent affinity reagents by in vitro selection. Here, we report identification and characterization of a novel helical polypeptide with advantageous biophysical properties as a template for construction of phage display libraries. A three-helix bundle structure, based on Measles virus phosphoprotein P shows a very favourable stability and solubility profile. We designed, constructed and characterized six different types of phage display libraries based on the proposed template. Their functional size of over 109 independent clones, balanced codon bias and decent display level are key parameters attesting to the quality and utility of the libraries. The new libraries are a promising tool for isolation of high affinity binders based on a small helical scaffold which could become a convenient alternative to antibodies

Characterization of a Single-Stranded DNA-Binding-Like Protein from Nanoarchaeum equitans--A Nucleic Acid Binding Protein with Broad Substrate Specificity.

SSB (single-stranded DNA-binding) proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis.This paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein). This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity). The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7 ± 1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100 °C and melting temperature (T(m)) is 100.2 °C as shown by differential scanning calorimetry (DSC) analysis.NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids

Sedimentation analysis of <i>Neq</i>SSB-like (A) and standard proteins (B).

<p>The proteins were analyzed on a 12% polyacrylamide gel. Lane M: Unstained Protein Weight Marker (Fermentas, Lithuania), with the molecular mass of proteins marked. Lane 1–19: fraction number. 50 μl of 150 μM <i>Neq</i>SSB-like and the corresponding amounts of standard proteins were centrifuged in linear 15 to 30% (w/v) glycerol gradients, as described in the “Methods”. The fractions with proteins were analyzed by SDS-PAGE. The fractions at which the maximal amount of protein appears are shown by arrows in each panel. The standard proteins used are: L, lysozyme (14 kDa); CA, carbonic anhydrase (29 kDa); BSA, bovine serum albumin (66 kDa) and AD, alcohol dehydrogenase (150 kDa). The oligomerization state estimation of <i>Neq</i>SSB-like was made with these proteins.</p

Binding of <i>Neq</i>SSB-like to M13 ssDNA.

<p>Lanes 1–8 contain (0.07 pmol) of M13 ssDNA and 0, 3.5, 7, 14, 28, 56, 112 and 224 pmoles of <i>Neq</i>SSB-like, respectively.</p

Results of the analytical gel filtration of <i>Neq</i>SSB-like on the Superdex 75 10/300 GL column.

<p><b>A</b> The standard linear regression curve was generated by plotting the log of the molecular mass of calibration proteins against V_e/V₀ value, namely elution volume divided by void volume. The calibration proteins represented by black triangles include bovine albumin (66 kDa), ovalbumin (43 kDa), carbon anhydrase (29 kDa) and ribonuclease A (13 kDa). <i>Neq</i>SSB-like is represented by the black circle. The regression curve equation and coefficient of determination are shown. <b>B</b> Effects of <i>Neq</i>SSB-like protein concentrations on the elution profiles of gel filtration. The entire range of <i>Neq</i>SSB-like concentrations (12–236 μM) show an elution volume of 15.8 ml corresponding to the monomeric protein.</p

The multiple amino acid alignment.

<p><b>A</b> The multiple amino acid alignment of nucleic acid binding protein from <i>Nanoarchaeum equitans</i>, bacterial and craenarchaeal SSB proteins. <b>B</b> The multiple amino acid alignment of nucleic acid binding protein from <i>Nanoarchaeum equitans</i> and craenarchaeal SSB proteins. The alignments were performed by dividing the amino acids into six similarity groups: group 1 V, L, I, M, group 2 W, F, Y, group 3 E, D, group 4 K, R, group 5 Q, D, and group 6 S, T. The capital letters represent single amino acid codes. White fonts on black boxes represent 100% similarity, white fonts on grey boxes denote <80% similarity, and black fonts on grey boxes show <60% similarity. Abbreviations: NeqSSB—nucleic acid binding protein from <i>Nanoarchaeum equitans</i> Kin-4M, SsoSSB <i>Sulfolobus solfataricus</i> strain P2, SacSSB <i>Sulfolobus acidocaldarius</i> DSM 639, SmaSSB <i>Staphylothermus marinus</i> F1, DkeSSB <i>Desulfurococcus kamchatkensis</i> 1221n, EcoSSB <i>Escherichia coli</i> K12, TmaSSB <i>Thermotoga maritima</i> strain MSB8, and TteSSB3 <i>Thermoanerobacter tengcongensis</i> MB4. The W108 residue, important in base-stacking interactions is indicated in the panel B. The blue box indicates OB-fold region.</p

<i>Neq</i>SSB-like dsDNA and mRNA binding properties.

<p><b>A</b> Binding to 2.5 pmol of 100 bp PCR product. Lanes 1–6 contain 0, 10, 20, 40, 80 and 160 pmoles of <i>Neq</i>SSB-like, respectively. <b>B</b> Binding to 0.132 pmol of <i>Escherichia coli</i> genomic DNA. Lanes 1–7 contain 0, 10, 20, 40, 80, 160 and 320 pmoles of <i>Neq</i>SSB-like, respectively. <b>C</b> Binding to 0.2 pmol of pDONR201 plasmid DNA (4470 bp). Lanes 1–7 contain 0, 10, 20, 40, 80, 160 and 320 pmoles of <i>Neq</i>SSB-like, respectively. <b>D</b> Binding to 0.1 pmol of pDONR201 plasmid DNA + 0.05 pmol of linearized pDONR201 plasmid DNA. Lanes 1–7 contain 0, 10, 20, 40, 80, 160 and 320 pmoles of <i>Neq</i>SSB-like, respectively. <b>E</b> Control binding reaction with 0.2 pmol of pDONR201 plasmid DNA. Lanes 1–4 contain 0, 10, 20 and 40 pmoles of <i>Taq</i>SSB, respectively. <b>F</b> Binding to 980 ng of mRNA. Lanes 1–5 contain 0, 10, 20, 40, 80 pmoles of <i>Neq</i>SSB-like, respectively.</p

<i>Neq</i>SSB-like binding preferences.

<p>The reactions contained a fixed quantity of the sample DNA: 10 pmol of (dT)₇₆ and 2.5 pmol of 100 bp PCR product. Lane 1: (dT)₇₆ with 0 pmol of <i>Neq</i>SSB-like. Lane 2: 100 bp with 0 pmol of <i>Neq</i>SSB-like. Lane 3: (dT)₇₆ and 100 bp PCR product with 0 pmol of <i>Neq</i>SSB-like. Lanes 4–9 contain 10, 20, 40, 80, 160 and 320 pmoles of <i>Neq</i>SSB-like, respectively.</p

The expression and purification of <i>Neq</i>SSB-like from <i>E</i>. <i>coli</i> TOP10F’+pBAD/NeqSSBHT.

<p>The proteins were analyzed on a 12% polyacrylamide gel. Lane M: Unstained Protein Weight Marker (Fermentas, Lithuania), with the molecular mass of proteins marked. Lane 1: soluble protein cell extracts after arabinose induction of protein expression (10 μl). Lane 2: <i>Neq</i>SSB-like after the Ni²⁺-affinity chromatography step (10 μl). Lane 3: <i>Neq</i>SSB-like after His-tag cleavage with TEV protease (10 μl). Lane 4: <i>Neq</i>SSB-like after chromatography on an ssDNA-cellulose column (10 μl).</p

Interaction analysis of <i>Neq</i>SSB-like (A) with ssDNA and dsDNA (B).

<p>Different concentrations of the protein were injected with a flow rate of 30 μl/min on a streptavidin chip coated with ssDNA 60-mer and dsDNA 60-mer on separate flow channels. A flow cell with streptavidin was used as a reference. After each injection the chip was regenerated with 0.01% SDS. The different colors of the sensograms represent the concentrations of the <i>Neq</i>SSB-like injected. Solid lines state for fitted curves. The data were fitted in accordance with the Langmuir model and using BiaEval 3.0 software.</p