Extremophilic 50S Ribosomal RNA-Binding Protein L35Ae as a Basis for Engineering of an Alternative Protein Scaffold

Abstract

Due to their remarkably high structural stability, proteins from extremophiles are particularly useful in numerous biological applications. Their utility as alternative protein scaffolds could be especially valuable in small antibody mimetic engineering. These artificial binding proteins occupy a specific niche between antibodies and low molecular weight substances, paving the way for development of innovative approaches in therapeutics, diagnostics, and reagent use. Here, the 50S ribosomal RNA-binding protein L35Ae from the extremophilic archaea Pyrococcus horikoshii has been probed for its potential to serve as a backbone in alternative scaffold engineering. The recombinant wild type L35Ae has a native-like secondary structure, extreme thermal stability (mid-transition temperature of 90°C) and a moderate resistance to the denaturation by guanidine hydrochloride (half-transition at 2.6 M). Chemical crosslinking and dynamic light scattering data revealed that the wild type L35Ae protein has a propensity for multimerization and aggregation correlating with its non-specific binding to a model cell surface of HEK293 cells, as evidenced by flow cytometry. To suppress these negative features, a 10-amino acid mutant (called L35Ae 10X) was designed, which lacks the interaction with HEK293 cells, is less susceptible to aggregation, and maintains native-like secondary structure and thermal stability. However, L35Ae 10X also shows lowered resistance to guanidine hydrochloride (half-transition at 2.0M) and is more prone to oligomerization. This investigation of an extremophile protein’s scaffolding potential demonstrates that lowered resistance to charged chemical denaturants and increased propensity to multimerization may limit the utility of extremophile proteins as alternative scaffolds.

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

“…The codon optimized for expression in E . coli gene of L35Ae 10X [30] with randomized regions coding the CDR-like loops 1–3 was assembled by overlap extension PCRs, using the primers listed in Table 1 (synthesized by Evrogen, Moscow, Russia).…”

“…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 Nco I and Not I 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 ’).…”

“…Indeed, Sso7d and Sac7d proteins from thermoacidophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius , respectively, have been successfully used for engineering of specific binders [25–28]. We have recently shown that 10X mutant (Fig 1A) of 50S ribosomal protein L35Ae from hyperthermophilic archaea Pyrococcus horikoshii [29] has several features favoring its use as a protein scaffold [30]: small size (88 residues), lack of disulfides, high thermal stability (mid-transition temperature of 90°C), efficient bacterial production (60 mg of protein per liter of cell culture), lack of non-specific binding to model human embryonic kidney 293 cells (HEK293), and the presence of three nearby loops closely resembling complementarity determining regions (CDRs) of immunoglobulins as judged from tertiary structure of L35Ae from P . furiosus (Fig 2A and 2C).…”

“…furiosus reveals a six-stranded antiparallel β-barrel with a short α-helix located between strands β2 and β3 with CDR-like loops between strands β1 and β2, β3 and β4, β5 and β6 (referred to as loops 1, 2 and 3, respectively—Figs 1 and 2) forming an extended nearly flat surface potentially suited for target recognition. The proposed on the basis of molecular modelling tRNA-binding site of L35Ae (involves strands β1, β2 and β5 [34]–Fig 2A) was impaired in L35Ae 10X mutant by suppression of its excessive positive charge (Fig 1A), which eliminated L35Ae interaction with surface of HEK293 cells [30]. …”

“…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.…”