Rapid and Direct VHH and Target Identification by Staphylococcal Surface Display Libraries
Abstract
:1. Introduction
2. Results
2.1. Expression and Attachment of Single-Domain Antigen-Binding Fragments from Camelid Heavy Chain-Only Antibodies (VHH) to the Peptidoglycan in S. aureus
2.2. Quantification of VHH Surface Exposure and Immunoprecipitation (IP) Capacity
2.3. Detection of GFP-Tagged Proteins with Low Expression Levels in Cell Lysates
2.4. Release of the Displayed VHH by the Endogenous Staphylococcal SrtA Enzyme
2.5. Direct Target Identification by Combined Staphylococcal Surface Display and IP
2.6. Target Verification and Characterization of VHH Identified by Staphylococcal Surface Display
3. Discussion
4. Materials and Methods
4.1. Generation and Mass Spectrometry of the pSA-VHH-SPAXrc Shuttle Vector Construct
4.2. Detection of Nitrocellulose Spotted S. aureus
4.3. Flow Cytometry and Fluorescence-Activated Cell Sorting (FACS) of S. aureus
4.4. IP with S. aureus
4.5. Fixation of S. aureus
4.6. Maintenance of C. elegans Strains and Lysate Preparation
4.7. GFP Western Blotting
4.8. VHH Release from S. aureus
4.9. Specificity and Competition Enzyme-Linked Immunosorbent Assay (ELISA) and Fast Protein Liquid Chromatography (FPLC)
Supplementary Materials:
Acknowledgments
Conflicts of Interest
Abbreviations
CDR | complementarity-determining region(s) |
CMFDA | 5-chloromethylfluorescein diacetate (CellTracker Green) |
enh | VHH specific for GFP (enhancer) |
FA | formaldehyde |
FACS | fluorescence-activated cell sorting |
FR | framework region(s) |
GFP | green fluorescent protein |
HA | hemagglutinin |
HRP | horseradish peroxidase |
IP | immunoprecipitation |
KO | knockout |
M1 | matrix protein 1 |
MHCII | major histocompatibility complex class II |
NP | nucleoprotein |
PBS | phosphate buffered saline |
PBSB | PBS, 2% BSA |
PBSI | PBS, 2% ICS |
PBSMT | PBS, 4% milk, 0.1% Tween-20 |
PE | phycoerythrin |
scFv | single-chain variable fragment(s) |
SMM | 0.5 M sucrose, 20 mM maleate, 20 mM MgCl2 (pH 6.5) |
SrtA | staphylococcal housekeeping sortase A |
TAMRA | 5-carboxytetramethylrhodamine |
TBSMT | Tris buffered saline, 5% milk, 0.1% Tween-20 |
VHH | single-domain antigen recognition fragment(s) from camelid heavy chain-only antibodies |
WT | wild type |
Xc | staphylococcal protein A constant region (amino acids 421–516) |
Xr | staphylococcal protein A repetitive region (amino acids 324–420) |
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Cavallari, M. Rapid and Direct VHH and Target Identification by Staphylococcal Surface Display Libraries. Int. J. Mol. Sci. 2017, 18, 1507. https://doi.org/10.3390/ijms18071507
Cavallari M. Rapid and Direct VHH and Target Identification by Staphylococcal Surface Display Libraries. International Journal of Molecular Sciences. 2017; 18(7):1507. https://doi.org/10.3390/ijms18071507
Chicago/Turabian StyleCavallari, Marco. 2017. "Rapid and Direct VHH and Target Identification by Staphylococcal Surface Display Libraries" International Journal of Molecular Sciences 18, no. 7: 1507. https://doi.org/10.3390/ijms18071507