A Novel Nanobody Scaffold Optimized for Bacterial Expression and Suitable for the Construction of Ribosome Display Libraries.
Amino Acid Sequence
/ genetics
Animals
Antibody Affinity
/ genetics
Camelus
/ genetics
Cloning, Molecular
Directed Molecular Evolution
Escherichia coli
/ genetics
Gene Expression
Peptide Library
Protein Engineering
Recombinant Fusion Proteins
/ genetics
Ribosomes
/ metabolism
Single-Domain Antibodies
/ genetics
Library
Nanobody
Ribosome display
Scaffold
VHH
Journal
Molecular biotechnology
ISSN: 1559-0305
Titre abrégé: Mol Biotechnol
Pays: Switzerland
ID NLM: 9423533
Informations de publication
Date de publication:
Jan 2020
Jan 2020
Historique:
pubmed:
14
11
2019
medline:
10
6
2020
entrez:
14
11
2019
Statut:
ppublish
Résumé
Single-domain antigen-binding fragments of camelid antibodies, known as VHHs or nanobodies, are widely used affinity reagents. However, their production involving animal immunization is time- and resource-intensive. Starting from a sequence dataset of llama VHHs, we designed a novel scaffold, based on conserved framework sequences, suitable for bacterial nanobody expression and synthetic library construction. The consensus scaffold was validated by grafting the CDRs from two known nanobodies. While maintaining their binding properties, the two chimeric nanobodies showed higher levels of expression and solubility in E. coli when compared to the corresponding wild types. A proof-of-concept synthetic combinatorial library, suitable for ribosome display (RD) selection, was obtained by encoding three randomized complementarity determining regions within the consensus framework. The library, made of linear DNA fragments, has an estimated complexity of > 10
Identifiants
pubmed: 31720928
doi: 10.1007/s12033-019-00224-z
pii: 10.1007/s12033-019-00224-z
doi:
Substances chimiques
Peptide Library
0
Recombinant Fusion Proteins
0
Single-Domain Antibodies
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
43-55Subventions
Organisme : Regione Emilia-Romagna
ID : PRUa1RI-2012-006
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