Tracking molecular recognition at the atomic level with a new protein scaffold based on the OB-fold.
The OB-fold is a small, versatile single-domain protein binding module that occurs in all forms of life, where it binds protein, carbohydrate, nucleic acid and small-molecule ligands. We have exploited this natural plasticity to engineer a new class of non-immunoglobulin alternatives to antibodies w...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2014-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3896448?pdf=render |
| _version_ | 1818020861371744256 |
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| author | John D Steemson Matthias Baake Jasna Rakonjac Vickery L Arcus Mark T Liddament |
| author_facet | John D Steemson Matthias Baake Jasna Rakonjac Vickery L Arcus Mark T Liddament |
| author_sort | John D Steemson |
| collection | DOAJ |
| description | The OB-fold is a small, versatile single-domain protein binding module that occurs in all forms of life, where it binds protein, carbohydrate, nucleic acid and small-molecule ligands. We have exploited this natural plasticity to engineer a new class of non-immunoglobulin alternatives to antibodies with unique structural and biophysical characteristics. We present here the engineering of the OB-fold anticodon recognition domain from aspartyl tRNA synthetase taken from the thermophile Pyrobaculum aerophilum. For this single-domain scaffold we have coined the term OBody. Starting from a naïve combinatorial library, we engineered an OBody with 3 nM affinity for hen egg-white lysozyme, by optimising the affinity of a naïve OBody 11,700-fold over several affinity maturation steps, using phage display. At each maturation step a crystal structure of the engineered OBody in complex with hen egg-white lysozyme was determined, showing binding elements in atomic detail. These structures have given us an unprecedented insight into the directed evolution of affinity for a single antigen on the molecular scale. The engineered OBodies retain the high thermal stability of the parental OB-fold despite mutation of up to 22% of their residues. They can be expressed in soluble form and also purified from bacteria at high yields. They also lack disulfide bonds. These data demonstrate the potential of OBodies as a new scaffold for the engineering of specific binding reagents and provide a platform for further development of future OBody-based applications. |
| first_indexed | 2024-04-14T08:11:06Z |
| format | Article |
| id | doaj.art-757fb2560a574efbaffec3c0fb00b9b3 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-14T08:11:06Z |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-757fb2560a574efbaffec3c0fb00b9b32022-12-22T02:04:33ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-0191e8605010.1371/journal.pone.0086050Tracking molecular recognition at the atomic level with a new protein scaffold based on the OB-fold.John D SteemsonMatthias BaakeJasna RakonjacVickery L ArcusMark T LiddamentThe OB-fold is a small, versatile single-domain protein binding module that occurs in all forms of life, where it binds protein, carbohydrate, nucleic acid and small-molecule ligands. We have exploited this natural plasticity to engineer a new class of non-immunoglobulin alternatives to antibodies with unique structural and biophysical characteristics. We present here the engineering of the OB-fold anticodon recognition domain from aspartyl tRNA synthetase taken from the thermophile Pyrobaculum aerophilum. For this single-domain scaffold we have coined the term OBody. Starting from a naïve combinatorial library, we engineered an OBody with 3 nM affinity for hen egg-white lysozyme, by optimising the affinity of a naïve OBody 11,700-fold over several affinity maturation steps, using phage display. At each maturation step a crystal structure of the engineered OBody in complex with hen egg-white lysozyme was determined, showing binding elements in atomic detail. These structures have given us an unprecedented insight into the directed evolution of affinity for a single antigen on the molecular scale. The engineered OBodies retain the high thermal stability of the parental OB-fold despite mutation of up to 22% of their residues. They can be expressed in soluble form and also purified from bacteria at high yields. They also lack disulfide bonds. These data demonstrate the potential of OBodies as a new scaffold for the engineering of specific binding reagents and provide a platform for further development of future OBody-based applications.http://europepmc.org/articles/PMC3896448?pdf=render |
| spellingShingle | John D Steemson Matthias Baake Jasna Rakonjac Vickery L Arcus Mark T Liddament Tracking molecular recognition at the atomic level with a new protein scaffold based on the OB-fold. PLoS ONE |
| title | Tracking molecular recognition at the atomic level with a new protein scaffold based on the OB-fold. |
| title_full | Tracking molecular recognition at the atomic level with a new protein scaffold based on the OB-fold. |
| title_fullStr | Tracking molecular recognition at the atomic level with a new protein scaffold based on the OB-fold. |
| title_full_unstemmed | Tracking molecular recognition at the atomic level with a new protein scaffold based on the OB-fold. |
| title_short | Tracking molecular recognition at the atomic level with a new protein scaffold based on the OB-fold. |
| title_sort | tracking molecular recognition at the atomic level with a new protein scaffold based on the ob fold |
| url | http://europepmc.org/articles/PMC3896448?pdf=render |
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