Biopanning of Phage Displayed Peptide Libraries for the Isolation of Cell-Specific Ligands

McGuire, Michael J.; Li, Shunzi; Brown, Kathlynn C.

doi:10.1007/978-1-60327-569-9_18

Cited by 48 publications

(44 citation statements)

References 16 publications

(20 reference statements)

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“…Phage display libraries are powerful tools to obtain specific ligands against small molecules, cells, and even tissues (31,(55)(56)(57)(58). Previously, this method has been used to identify M celltargeting ligands using rat PPs, although the identified peptides showed high binding affinity only to mucosa and Caco-2 cells (46).…”

Section: Discussionmentioning

confidence: 99%

The M Cell-Targeting Ligand Promotes Antigen Delivery and Induces Antigen-Specific Immune Responses in Mucosal Vaccination

Kim¹,

Seo²,

Kim³

et al. 2010

The Journal of Immunology

114

100

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Oral mucosal immunization can induce protective immunity in both systemic compartments and the mucosa. Successful mucosal immunization depends on Ag delivery to the mucosal immune induction site. The high transcytotic activity of M cells within the mucosa makes these cells attractive targets for mucosal Ag delivery, although it remains unclear whether delivery of Ag to M cells only can guarantee the induction of effective immune responses. In this study, we evaluated the ability of an M cell-targeting ligand with adjuvant activity to induce immunity against ligand-fused Ag. We selected M cell-targeting ligands through biopanning of a phage display library against differentiated in vitro M-like cells and produced the recombinant Ags fused to the selected ligands using the model Ag. One of the selected peptide ligands, Co1, promoted the binding of ligand-fused Ag to mouse Peyer’s patch M cells and human M-like cells that had been defined by binding with the M cell-specific and anti-GP2 Abs. In addition, Co1 ligand enhanced the uptake of fused Ag by immunogenic tissue in an ex vivo loop assay and in vivo oral administration experiments. After oral administration, the ligand-fused Ag enhanced immune responses against the fused Ag compared with those of the control Ag without ligand. In addition, this use of the ligand supported a skewed Th2-type immune response against the fused Ag. Collectively, these results suggest that the ligand selected through biopanning against cultured M-like cells could be used as an adjuvant for targeted Ag delivery into the mucosal immune system to enhance immune induction.

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Section: Discussionmentioning

confidence: 99%

The M Cell-Targeting Ligand Promotes Antigen Delivery and Induces Antigen-Specific Immune Responses in Mucosal Vaccination

Kim¹,

Seo²,

Kim³

et al. 2010

The Journal of Immunology

114

100

View full text Add to dashboard Cite

show abstract

“…The library presents approx. 1 x 10 8 different types of random 20-mer peptide sequences, and was kindly provided to us by Dr. Kathlynn Brown's laboratory at UT Southwestern Medical Center 26 . The phage infection into the E. coli K91 makes the bacteria resistant to 12 μg/ml tetracycline.…”

Section: Screening For Conformation-specific Ligands Against Channelsmentioning

confidence: 99%

“…A detailed procedure for the bacterial culture, the amplification and titering of the phages and the sequencing of the phage colonies has been described by McGuire, et al 26 We will give a short description of the basic operations in the next section. The readers can find more details in the McGuire paper.…”

Section: Screening For Conformation-specific Ligands Against Channelsmentioning

confidence: 99%

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Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

Lee

Zheng

Shi

et al. 2013

JoVE

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To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of welldefined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels. Video LinkThe video component of this article can be found at http://www.jove.com/video/50436/ IntroductionCells exchange materials and information with their environment through the functions of specific membrane proteins 1 . Membrane proteins in cell membranes function as pumps, channels, receptors, intramembrane enzymes, linkers and structural supporters across membranes. Mutations that affect the membrane proteins have been related to many human diseases. In fact, many membrane proteins have been the primary drug targets because they are important and easily accessible in cell membranes. It is therefore very important to understand the structure and function of various membrane proteins in membranes, and make it possible to devise novel methods to alleviate the detrimental effects from the mutant proteins in human diseases.Lipids surround all membrane proteins integrated in bilayers 2,3 . In eukaryotic membranes, the various different types of lipids are known to be organized into microdomains 4, 5 . Many membrane proteins were shown to be distributed among these microdomains as well as the bulky fluid phase of membranes 3,6 . The mechanism underlying the organization of the microdomains and the delivery of membrane proteins into them and the physiological significance of such distributions are clearly important but remain poorly understood. One major technical difficulty in studying the lipid effects on membrane proteins is the reliable reconstitution of biochemically purified membrane proteins int...

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“…A 20-mer peptide is displayed on the pIII coat protein encoded by ligation of random NNK oligonucleotides (14,15). Because of the library's design, it contains the rare AGG codon for arginine (Arg), whose tRNA is encoded by the pRARE plasmid.…”

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confidence: 99%

Introduction of Plasmid Encoding for Rare tRNAs Reduces Amplification Bias in Phage Display Biopanning

2015

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Amplification bias is a major hurdle in phage display protocols because it imparts additional, unintended selection pressure beyond binding to the desired target. One potential source of amplification bias is the inherent lack of codon optimization that occurs within phage display libraries. Here we present a method that reduces amplification bias by addition of a plasmid that encodes six low abundance tRNAs into K91 Escherichia coli. This new strain, termed K91+, is used to amplify phage during the selection process. We demonstrate the importance of rare codon usage in phage production, and our method produced an overall increase in uniformity of phage production in a random library. Both of these variables are improved in E. coli K91+ compared with the parental K91 strain. This simple solution, requiring only a commercially available plasmid and an additional antibiotic, can reduce amplification bias in phage display protocols.

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Biopanning of Phage Displayed Peptide Libraries for the Isolation of Cell-Specific Ligands

Cited by 48 publications

References 16 publications

The M Cell-Targeting Ligand Promotes Antigen Delivery and Induces Antigen-Specific Immune Responses in Mucosal Vaccination

The M Cell-Targeting Ligand Promotes Antigen Delivery and Induces Antigen-Specific Immune Responses in Mucosal Vaccination

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

Introduction of Plasmid Encoding for Rare tRNAs Reduces Amplification Bias in Phage Display Biopanning

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