Engineered high-affinity nanobodies recognizing staphylococcal Protein A and suitable for native isolation of protein complexes

Fridy, Peter C.; Thompson, Matthew; Ketaren, Natalia E.; Rout, Michael P.

doi:10.1016/j.ab.2015.02.013

Cited by 18 publications

(11 citation statements)

References 26 publications

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“…Complete or near-complete sequence conservation at some V H H positions (Gly15, Ser17, Arg66, Ser70 and Gln81) among both SpA-binding V H Hs and non-SpA-binding V H Hs prevented the assessment of their importance. In agreement with the conclusions of structural studies [ 25 ] and a previous mutagenesis study [ 57 ], the salt bridge formed between the Asp residue at position 36 of SpA domain D and the conserved Arg residue at V H H FR1 position 19 was indispensable, as its replacement with Lys, Ser or Thr abrogated SpA binding. Similarly, replacement of core Gly65 with negatively charged Asp, or of core Asn82a with Asp or Ser, had a destructive effect on SpA binding; the latter result is consistent with a previous mutagenesis study [ 57 ] which found that substitution of Asn82a with Ala abrogated SpA binding.…”

Section: Resultssupporting

confidence: 90%

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A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies

et al. 2016

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Staphylococcal protein A (SpA) and streptococcal protein G (SpG) affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs) in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs) are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding VHHs. We show that SpA binding of VHHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species.

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Section: Resultssupporting

confidence: 90%

“… Residues in brackets are tolerated but result in some loss of binding a Data from Fridy et al . [ 57 ] …”

Section: Resultsmentioning

confidence: 99%

A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies

et al. 2016

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“…This is appropriate for an identification, but hardly for any further downstream structural or functional analysis of the purified target proteins. As a workaround, a native isolation of protein A-tagged protein complexes using a specific nanobody modified with a dithiothreitol (DTT)-cleavable crosslinker was recently reported ( Fridy et al, 2015 ). However, the achievable yield was modest, as most of the isolated complexes resisted release.…”

Section: Introductionmentioning

confidence: 99%

Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation

Pleiner

Bates

Trakhanov

et al. 2015

eLife

202

258

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Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with <2 nm epitope-label displacement. For this, we introduced cysteines at specific positions in the nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks – each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target.DOI: http://dx.doi.org/10.7554/eLife.11349.001

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“…The fold prediction software Phyre2 was used to model V H H against Urease (Hoseinpoor et al, 2014). The popular i-TASSER web server was used for V H H designed against PrA (ProteinA) (Fridy et al, 2015) and HCV Non-structural protein NS3/4A (Jittavisutthikul et al, 2015). Raptor-X, another threading-based server, was used to model V H H against adenylate cyclase-hemolysin toxin and the repeats in toxin (CyaA-RTX protein) (Malik et al, 2016) subdomains (see Supplemental Information 1).…”

Section: (B) Other Generic 3d Prediction Approachesmentioning

confidence: 99%

Discrete analysis of camelid variable domains: sequences, structures, and in-silico structure prediction

Vattekatte

Shinada

Narwani

et al. 2020

PeerJ

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Antigen binding by antibodies requires precise orientation of the complementarity- determining region (CDR) loops in the variable domain to establish the correct contact surface. Members of the family Camelidae have a modified form of immunoglobulin gamma (IgG) with only heavy chains, called Heavy Chain only Antibodies (HCAb). Antigen binding in HCAbs is mediated by only three CDR loops from the single variable domain (VHH) at the N-terminus of each heavy chain. This feature of the VHH, along with their other important features, e.g., easy expression, small size, thermo-stability and hydrophilicity, made them promising candidates for therapeutics and diagnostics. Thus, to design better VHH domains, it is important to thoroughly understand their sequence and structure characteristics and relationship. In this study, sequence characteristics of VHH domains have been analysed in depth, along with their structural features using innovative approaches, namely a structural alphabet. An elaborate summary of various studies proposing structural models of VHH domains showed diversity in the algorithms used. Finally, a case study to elucidate the differences in structural models from single and multiple templates is presented. In this case study, along with the above-mentioned aspects of VHH, an exciting view of various factors in structure prediction of VHH, like template framework selection, is also discussed.

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Engineered high-affinity nanobodies recognizing staphylococcal Protein A and suitable for native isolation of protein complexes

Cited by 18 publications

References 26 publications

A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies

A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies

Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation

Discrete analysis of camelid variable domains: sequences, structures, and in-silico structure prediction

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