Derivative of Extremophilic 50S Ribosomal Protein L35Ae as an Alternative Protein Scaffold

Abstract

Small antibody mimetics, or alternative binding proteins (ABPs), extend and complement antibody functionality with numerous applications in research, diagnostics and therapeutics. Given the superiority of ABPs, the last two decades have witnessed development of dozens of alternative protein scaffolds (APSs) for the design of ABPs. Proteins from extremophiles with their high structural stability are especially favorable for APS design. Here, a 10X mutant of the 50S ribosomal protein L35Ae from hyperthermophilic archaea Pyrococcus horikoshii has been probed as an APS. A phage display library of L35Ae 10X was generated by randomization of its three CDR-like loop regions (repertoire size of 2×108). Two L35Ae 10X variants specific to a model target, the hen egg-white lysozyme (HEL), were isolated from the resulting library using phage display. The affinity of these variants (L4 and L7) to HEL ranges from 0.10 μM to 1.6 μM, according to surface plasmon resonance data. While L4 has 1-2 orders of magnitude lower affinity to HEL homologue, bovine α-lactalbumin (BLA), L7 is equally specific to HEL and BLA. The reference L35Ae 10X is non-specific to both HEL and BLA. L4 and L7 are more resistant to denaturation by guanidine hydrochloride compared to the reference L35Ae 10X (mid-transition concentration is higher by 0.1-0.5 M). Chemical crosslinking experiments reveal an increased propensity of L4 and L7 to multimerization. Overall, the CDR-like loop regions of L35Ae 10X represent a proper interface for generation of functional ABPs. Hence, L35Ae is shown to extend the growing family of protein scaffolds dedicated to the design of novel binding proteins.

Fig 1. Amino acid sequences for the recombinant forms of L35Ae from P. horikoshii used in this study.

Secondary structure elements of L35Ae from P. furiosus are indicated (refer to PDB entry 2lp6 [34]): β-sheets 1–6, CDR-like loops 1–3, α-helix (green). The residues affected by randomization are shown in color. (A) The amino acid sequences of rWT L35Ae and its 10X mutant [30]. The residues differing between 10X and rWT L35Ae are indicated using bold font. Two 10X variants were used in the phage display library, which contain a loop 2 of original length (a) or elongated by three residues (b). (B) The amino acid sequences of L35Ae 10X with C-terminal GLE sequence replaced by myc tag (‘10X-myc’) and those for HEL-specific binders L4 and L7, isolated from the phage display library of L35Ae 10X. The regions subjected to randomization are indicated in pink.

Fig 2. Tertiary structures of 50S ribosomal protein L35Ae from Pyrococcus furiosus (sequence identity to L35Ae from P. horikoshii is 93%; PDB entry 2lp6 [34]) and heavy chain variable region of 1F1 antibody to the influenza A virus hemagglutinin (PDB entry 4gxv [39]).

The figure was created using ICM Browser v.3.7-3b (MolSoft L.L.C.) software. (A) The β-sheets from 1 to 6 and CDR-like loops 1 to 3 are indicated. The residues randomized in the phage display library of L35Ae 10X (Fig 1) are shown using wire representation (backbone is indicated in red). (C) Wire (left) and space-filling (right) representations of top view of the L35Ae structure shown in panel A.

Fig 3. A schematic map of the pSFR1 phagemid (Antherix, Pushchino, Russia) used for phage display of L35Ae 10X.

The phagemid is based on pSMART LC Amp vector (Lucigen^®). Ampicillin resistance of a bacterial host is ensured by the β-lactamase gene, ‘bla-gene’. The gene of the 10X mutant of L35Ae from P. horikoshii was codon optimized for expression in E. coli [30], subjected to randomization of the regions coding the CDR-like loops 1–3 (Fig 1A, S1 Fig) and cloned between the NcoI and NotI restriction sites. The L35Ae gene is followed by a myc tag (‘Myc-tag’), a 6×His tag (‘His-tag’) and the gene of the attachment protein G3P from Enterobacteria phage M13 (‘G3P’). The translated chimera of L35Ae 10X-myc (Fig 1B) and G3P is secreted due to the presence of a N-terminal pelB leader sequence (‘pLB-leader’).

Fig 4. Phage display library enrichment for L35Ae 10X variants specific to HEL during the selection rounds.

Specificity to HEL for the polyclonal phage particles after rounds 1–3 was estimated by indirect ELISA. HEL was nonspecifically immobilized on medium-binding ELISA microplate (blocking with 0.5% MPBST); the tightly bound phage particles were detected by absorbance at 493 nm using horseradish peroxidase conjugated to anti-M13 monoclonal antibody and o-phenylenediamine as the substrate. The background ELISA signal was measured using the same protocol, but without HEL immobilization.

Fig 5. Resistance of anti-HEL binders L4/L7 and L35Ae 10X-myc to GuHCl-induced unfolding monitored by intrinsic fluorescence emission spectroscopy.

Excitation wavelength was 280 nm. The normalized fluorescence intensity at 314 nm (F_{314 nm}) and fluorescence spectrum maximum position (λ_max) are shown. Protein concentration was 3 μM. 20 mM H₃BO₃, 300 mM NaCl, pH 8.8 buffer; 25°C. The dashed curves are theoretical fits to the experimental data using the Boltzmann function (Eq (1)). The resulting [GuHCl]_1/2 values estimated from the F_{314 nm} data: (1.54±0.09) M, (2.01±0.02) M and (1.63±0.10) M for L35Ae 10X-myc, L4 and L7, respectively.

Fig 6. Interaction kinetics for anti-HEL binders L4/L7 with HEL (panels A, B, respectively) or BLA (panels C, D, respectively) at 25°C (PBST buffer), monitored by SPR spectroscopy using HEL/BLA as a ligand.

Gray curves are experimental, while black curves are theoretical, calculated according to the heterogeneous ligand model (2) (see Table 3 for the fitting parameters). The analyte concentrations are indicated on the curves in μM.