Orthogonal Synthetic Zippers as Protein Scaffolds

Protein scaffolds have proven useful for co-localization of enzymes, providing control over stoichiometry and leading to higher local enzyme concentrations, which have led to improved product formation. To broaden their usefulness, it is necessary to have a wide choice of building blocks to mix and match for scaffold generation. Ideally, the scaffold building blocks should function at any location within the scaffold and have high affinity interactions with their binding partners. We examined the utility of orthogonal synthetic coiled coils (zippers) as scaffold components. The orthogonal zippers are coiled coil domains that form heterodimers only with their specific partner and not with other zipper domains. Focusing on two orthogonal zipper pairs, we demonstrated that they are able to function on either end or in the middle of a multiblock assembly. Surface plasmon resonance was employed to assess the binding kinetics of zipper pairs placed at the start, middle, or end of a construct. Size-exclusion chromatography was used to demonstrate the ability of a scaffold with two zipper domains to bind their partners simultaneously. We then expanded the study to examine the binding kinetics and cross-reactivities of three additional zipper pairs. By validating the affinities and specificities of synthetic zipper pairs, we demonstrated the potential for zipper domains to provide an expanded library of scaffolding parts for tethering enzymes in complex pathways for synthetic biology applications

Sequences of L1 binding sdAb.

<p>The alignment shows the sequences of L1 binders isolated in sandwich-style selection. Three families, based on homology within the CDR regions were isolated. CDR regions are underlined. Red denotes positions where the amino acid is 90% conserved in the compared sequences, blue indicates low consensus (50%) and black are not conserved. The most dominant family (L1-H7 family) showed some variation in amino acid sequences of the framework regions that showed an impact of binding affinities and thermal stability. Arrows mark the positions of the cysteine substitutions in the L1-G2+ construct.</p

Recombinant vaccinia virus proteins and antibodies from BEI resources.

<p>Recombinant vaccinia virus proteins and antibodies from BEI resources.</p

Specificity of L1 binding sdAbs.

<p>Direct binding assays were used to demonstrate target specificity for L1 binding antigens. Antigen was immobilized to magnetic beads and biotinylated (bt)- sdAbs (noted at the top of each graph) served as the tracers. Independent direct binding assays were performed different days; a representative data set is shown. The MAGPIX assays typically measures binding from at least 50 beads and the error of the mean fluorescence intensity (MFI) is ≤5%.</p

Amino acid alignments of vaccinia virus binding sdAbs.

<p>The representatives from each family that were chosen for further characterization are presented. Red denotes positions where the amino acid is 90% conserved in the compared sequences, blue indicates low consensus (50%) and black are not conserved. The three CDRs are underlined.</p

Melting temperatures of L1 binding sdAbs and mAbs.

<p>ND  =  not determined.</p><p>* Fluorescent melt assays performed in triplicate on at least 2 days. The triplicate measurements were typically identical within less than 0.2°C, and always within 0.6°C, while samples measured from different protein lots or on different days agreed within a degree. ** Most of the CD assays were performed on at least two different days. Values determined by CD agreed within a degree.</p><p>Melting temperatures of L1 binding sdAbs and mAbs.</p

Refolding of sdAb.

<p>Circular dichroism was used to monitor the secondary structure of purified sdAbs over a temperature range of 25–95°C. Representative plots are shown of clones H7 and H2 typifying the distinctly different refolding properties. Temperature is expressed in °C and the change in ellipticity is in milidegrees. CD experiments were performed in duplicate for most of the sdAbs; the observed Tm values agreed within a degree, and the refolding trends were the same among the replicate measurements.</p

Binding kinetics for sdAbs targeting the L1 antigen^*.

<p>* From binding to at least 3 L1-coated spots, reported as average (standard deviation).</p><p>Binding kinetics for sdAbs targeting the L1 antigen^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106263#nt101" target="_blank">*</a>}.</p

Increased melting temperature of L1-G2+.

<p>Fluorescence based melting assay showing melting of L1-G2 in blue and the mutant with the added disulfide, L1-G2+, in black. Melting temperature is determined from a plot of the first derivative of the melt curve. The measurements were performed in triplicate on two different days and showed identical results; for clarity only one replicate is shown.</p

Melting temperatures of vaccinia binding sdAbs.

<p>* Fluorescent melt assays performed in triplicate; the measurements were typically identical within less than 0.2°C, and always within 0.6°C.</p><p>Melting temperatures of vaccinia binding sdAbs.</p