Protease-resistant single-domain antibodies inhibit Campylobacter jejuni motility

Camelid heavy-chain antibody variable domains (VHHs) are emerging as potential antimicrobial reagents. We have engineered a previously isolated VHH (FlagV1M), which binds Campylobacter jejuni flagella, for greater thermal and proteolytic stability. Mutants of FlagV1M were obtained from an error-pron...

Full description

Saved in:
Bibliographic Details
Published inProtein engineering, design and selection Vol. 27; no. 6; pp. 191 - 198
Main Authors Hussack, Greg, Riazi, Ali, Ryan, Shannon, van Faassen, Henk, MacKenzie, Roger, Tanha, Jamshid, Arbabi-Ghahroudi, Mehdi
Format Journal Article
LanguageEnglish
Published England Oxford University Press 01.06.2014
Subjects
Amino Acid Sequence
Amino Acid Substitution
Anti-Bacterial Agents - chemistry
Anti-Bacterial Agents - pharmacology
Campylobacter jejuni
Campylobacter jejuni - drug effects
Campylobacter jejuni - physiology
Chymotrypsin - chemistry
Directed Molecular Evolution
Flagella - immunology
Flagellin - immunology
Hydrogen-Ion Concentration
Molecular Sequence Data
Pepsin A - chemistry
Protein Binding
Protein Denaturation
Proteolysis
Single-Chain Antibodies - chemistry
Single-Chain Antibodies - genetics
Single-Chain Antibodies - pharmacology
Trypsin - chemistry
H
motility
protein stability
V
protease resistance
VHH
C. jejuni
Online AccessGet full text

Cover

More Information
Summary:Camelid heavy-chain antibody variable domains (VHHs) are emerging as potential antimicrobial reagents. We have engineered a previously isolated VHH (FlagV1M), which binds Campylobacter jejuni flagella, for greater thermal and proteolytic stability. Mutants of FlagV1M were obtained from an error-prone polymerase chain reaction library that was panned in the presence of gastrointestinal (GI) proteases. Additional FlagV1M mutants were obtained through disulfide-bond engineering. Each approach produced VHHs with enhanced thermal stability and protease resistance. When the beneficial mutations from both approaches were combined, a hyperstabilized VHH was created with superior stability. The hyperstabilized VHH bound C. jejuni flagella with wild-type affinity and was capable of potently inhibiting C. jejuni motility in assays performed after sequential digestion with three major GI proteases, demonstrating the remarkable stability imparted to the VHH by combining our engineering approaches.
Bibliography:ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
ObjectType-Article-2
ObjectType-Feature-1
ISSN:1741-0126
1741-0134
DOI:10.1093/protein/gzu011