title: 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.
authors.affiliation: Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region, Russia.
authors.forename: Anna V
authors.initials: AV
authors.lastname: Lomonosova
authors.affiliation: Antherix, Pushchino, Moscow region, Russia.
authors.forename: Andrei B
authors.initials: AB
authors.lastname: Ulitin
authors.affiliation: Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region, Russia.
authors.forename: Alexei S
authors.initials: AS
authors.lastname: Kazakov
authors.affiliation: Antherix, Pushchino, Moscow region, Russia.|Biomirex Inc., Watertown, Massachusetts, United States of America.
authors.forename: Tajib A
authors.initials: TA
authors.lastname: Mirzabekov
authors.affiliation: Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region, Russia.
authors.forename: Eugene A
authors.initials: EA
authors.lastname: Permyakov
authors.affiliation: Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region, Russia.
authors.forename: Sergei E
authors.identifier: http://orcid.org/0000-0003-4086-3137
authors.initials: SE
authors.lastname: Permyakov
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D040761:Biomimetic Materials / Q000737:chemistry / Q000378:metabolism; D001709:Biotechnology; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002645:Chickens; D000071825:Extremophiles / Q000737:chemistry / Q000235:genetics; D007768:Lactalbumin / Q000378:metabolism; D008958:Models, Molecular; D009113:Muramidase / Q000378:metabolism; D019151:Peptide Library; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D043902:Pyrococcus horikoshii / Q000737:chemistry* / Q000235:genetics; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D002352:Carrier Proteins; D019151:Peptide Library; D012269:Ribosomal Proteins; D007768:Lactalbumin; C421864:hen egg lysozyme; D009113:Muramidase