Cargando…

Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands

Protein A affinity chromatography is widely used for the large-scale purification of antibodies because of its high yield, selectivity, and compatibility with NaOH sanitation. A general platform to produce robust affinity capture ligands for proteins beyond antibodies would improve bioprocessing eff...

Descripción completa

Detalles Bibliográficos
Autores principales: Suderman, Richard J., Gibson, Shane D., Strecker, Mary, Bonner, Amanda M., Chao, David M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10404686/
https://www.ncbi.nlm.nih.gov/pubmed/37315789
http://dx.doi.org/10.1016/j.jbc.2023.104910
_version_ 1785085351667171328
author Suderman, Richard J.
Gibson, Shane D.
Strecker, Mary
Bonner, Amanda M.
Chao, David M.
author_facet Suderman, Richard J.
Gibson, Shane D.
Strecker, Mary
Bonner, Amanda M.
Chao, David M.
author_sort Suderman, Richard J.
collection PubMed
description Protein A affinity chromatography is widely used for the large-scale purification of antibodies because of its high yield, selectivity, and compatibility with NaOH sanitation. A general platform to produce robust affinity capture ligands for proteins beyond antibodies would improve bioprocessing efficiency. We previously developed nanoCLAMPs (nano Clostridial Antibody Mimetic Proteins), a class of antibody mimetic proteins useful as lab-scale affinity capture reagents. This work describes a protein engineering campaign to develop a more robust nanoCLAMP scaffold compatible with harsh bioprocessing conditions. The campaign generated an improved scaffold with dramatically improved resistance to heat, proteases, and NaOH. To isolate additional nanoCLAMPs based on this scaffold, we constructed a randomized library of 1 × 10(10) clones and isolated binders to several targets. We then performed an in-depth characterization of nanoCLAMPs recognizing yeast SUMO, a fusion partner used for the purification of recombinant proteins. These second-generation nanoCLAMPs typically had a K(d) of <80 nM, a T(m) of >70 °C, and a t(1/2) in 0.1 mg/ml trypsin of >20 h. Affinity chromatography resins bearing these next-generation nanoCLAMPs enabled single-step purifications of SUMO fusions. Bound target proteins could be eluted at neutral or acidic pH. These affinity resins maintained binding capacity and selectivity over 20 purification cycles, each including 10 min of cleaning-in-place with 0.1 M NaOH, and remained functional after exposure to 100% DMF and autoclaving. The improved nanoCLAMP scaffold will enable the development of robust, high-performance affinity chromatography resins against a wide range of protein targets.
format Online
Article
Text
id pubmed-10404686
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher American Society for Biochemistry and Molecular Biology
record_format MEDLINE/PubMed
spelling pubmed-104046862023-08-08 Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands Suderman, Richard J. Gibson, Shane D. Strecker, Mary Bonner, Amanda M. Chao, David M. J Biol Chem Methods and Resources Protein A affinity chromatography is widely used for the large-scale purification of antibodies because of its high yield, selectivity, and compatibility with NaOH sanitation. A general platform to produce robust affinity capture ligands for proteins beyond antibodies would improve bioprocessing efficiency. We previously developed nanoCLAMPs (nano Clostridial Antibody Mimetic Proteins), a class of antibody mimetic proteins useful as lab-scale affinity capture reagents. This work describes a protein engineering campaign to develop a more robust nanoCLAMP scaffold compatible with harsh bioprocessing conditions. The campaign generated an improved scaffold with dramatically improved resistance to heat, proteases, and NaOH. To isolate additional nanoCLAMPs based on this scaffold, we constructed a randomized library of 1 × 10(10) clones and isolated binders to several targets. We then performed an in-depth characterization of nanoCLAMPs recognizing yeast SUMO, a fusion partner used for the purification of recombinant proteins. These second-generation nanoCLAMPs typically had a K(d) of <80 nM, a T(m) of >70 °C, and a t(1/2) in 0.1 mg/ml trypsin of >20 h. Affinity chromatography resins bearing these next-generation nanoCLAMPs enabled single-step purifications of SUMO fusions. Bound target proteins could be eluted at neutral or acidic pH. These affinity resins maintained binding capacity and selectivity over 20 purification cycles, each including 10 min of cleaning-in-place with 0.1 M NaOH, and remained functional after exposure to 100% DMF and autoclaving. The improved nanoCLAMP scaffold will enable the development of robust, high-performance affinity chromatography resins against a wide range of protein targets. American Society for Biochemistry and Molecular Biology 2023-06-12 /pmc/articles/PMC10404686/ /pubmed/37315789 http://dx.doi.org/10.1016/j.jbc.2023.104910 Text en © 2023 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Methods and Resources
Suderman, Richard J.
Gibson, Shane D.
Strecker, Mary
Bonner, Amanda M.
Chao, David M.
Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands
title Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands
title_full Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands
title_fullStr Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands
title_full_unstemmed Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands
title_short Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands
title_sort protein engineering of a nanoclamp antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands
topic Methods and Resources
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10404686/
https://www.ncbi.nlm.nih.gov/pubmed/37315789
http://dx.doi.org/10.1016/j.jbc.2023.104910
work_keys_str_mv AT sudermanrichardj proteinengineeringofananoclampantibodymimeticscaffoldasaplatformforproducingbioprocesscompatibleaffinitycaptureligands
AT gibsonshaned proteinengineeringofananoclampantibodymimeticscaffoldasaplatformforproducingbioprocesscompatibleaffinitycaptureligands
AT streckermary proteinengineeringofananoclampantibodymimeticscaffoldasaplatformforproducingbioprocesscompatibleaffinitycaptureligands
AT bonneramandam proteinengineeringofananoclampantibodymimeticscaffoldasaplatformforproducingbioprocesscompatibleaffinitycaptureligands
AT chaodavidm proteinengineeringofananoclampantibodymimeticscaffoldasaplatformforproducingbioprocesscompatibleaffinitycaptureligands