Name: Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity (Video) | JoVE Journal
Uploaded: 2018-05-01T14:30:00.000+00:00
Duration: 12 min 31 s
Description: Learn to construct a synthetic phage-displayed human antibody library with tailored diversity for therapeutic monoclonal antibody development. This method bypasses animal immunization and enables high-affinity antibody selection.

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The authors appreciate Dr. Frederic Fellouse from the Sidhu lab for critical comments on Kunkel&#39;s method based synthetic Fab phage library construction. The authors appreciate Mrs. Alevtina Pavlenco and other members from the Sidhu lab for valuable help of preparing high-efficiency electro-competent E. coli cells and high quality dU-ssDNA. This work was supported by National Natural Science Foundation of China (Grant No.: 81572698, 31771006) to DW and by ShanghaiTech University (Grant No.: F-0301-13-005) to Laboratory of Antibody Engineering.

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<li>Reichert, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antibodies+to+watch+in+2017%5BTitle%5D)+AND+%22MAbs%22%5BJournal%5D)">Antibodies to watch in 2017.</a> MAbs. 9 (2), 167-181 (2017).</li>
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<li>Sidhu, S. S., Fellouse, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthetic+therapeutic+antibodies%5BTitle%5D)+AND+%22Nat+Chem+Biol%22%5BJournal%5D)">Synthetic therapeutic antibodies.</a> Nat Chem Biol. 2 (12), 682-688 (2006).</li>
<li>Adams, J. J., Sidhu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthetic+antibody+technologies%5BTitle%5D)+AND+%22Curr+Opin+Struct+Biol%22%5BJournal%5D)">Synthetic antibody technologies.</a> Curr Opin Struct Biol. 24, 1-9 (2014).</li>
<li>Kunkel, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+and+efficient+site-specific+mutagenesis+without+phenotypic+selection%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Rapid and efficient site-specific mutagenesis without phenotypic selection.</a> Proc Natl Acad Sci U S A. 82 (2), 488-492 (1985).</li>
<li>Sidhu, S. S., Lowman, H. B., Cunningham, B. C., Wells, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phage+display+for+selection+of+novel+binding+peptides%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Phage display for selection of novel binding peptides.</a> Methods Enzymol. 328, 333-363 (2000).</li>
<li>Persson, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CDR-H3+diversity+is+not+required+for+antigen+recognition+by+synthetic+antibodies%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">CDR-H3 diversity is not required for antigen recognition by synthetic antibodies.</a> J Mol Biol. 425 (4), 803-811 (2013).</li>
<li>Lefranc, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IMGT+unique+numbering+for+immunoglobulin+and+T+cell+receptor+variable+domains+and+Ig+superfamily+V-like+domains%5BTitle%5D)+AND+%22Dev+Comp+Immunol%22%5BJournal%5D)">IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains.</a> Dev Comp Immunol. 27 (1), 55-77 (2003).</li>
<li>Graille, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complex+between+Peptostreptococcus+magnus+protein+L+and+a+human+antibody+reveals+structural+convergence+in+the+interaction+modes+of+Fab+binding+proteins%5BTitle%5D)+AND+%22Structure%22%5BJournal%5D)">Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins.</a> Structure. 9 (8), 679-687 (2001).</li>
<li>Graille, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crystal+structure+of+a+Staphylococcus+aureus+protein+A+domain+complexed+with+the+Fab+fragment+of+a+human+IgM+antibody%3A+structural+basis+for+recognition+of+B-cell+receptors+and+superantigen+activity%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: structural basis for recognition of B-cell receptors and superantigen activity.</a> Proc Natl Acad Sci U S A. 97 (10), 5399-5404 (2000).</li>
<li>Huang, R., Fang, P., Kay, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improvements+to+the+Kunkel+mutagenesis+protocol+for+constructing+primary+and+secondary+phage-display+libraries%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries.</a> Methods. 58 (1), 10-17 (2012).</li>
<li>Chen, G., Sidhu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Design+and+generation+of+synthetic+antibody+libraries+for+phage+display%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Design and generation of synthetic antibody libraries for phage display.</a> Methods Mol Biol. 1131, 113-131 (2014).</li>
<li>Padlan, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anatomy+of+the+antibody+molecule%5BTitle%5D)+AND+%22Mol+Immunol%22%5BJournal%5D)">Anatomy of the antibody molecule.</a> Mol Immunol. 31 (3), 169-217 (1994).</li>
<li>Virnekas, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trinucleotide+phosphoramidites%3A+ideal+reagents+for+the+synthesis+of+mixed+oligonucleotides+for+random+mutagenesis%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis.</a> Nucleic Acids Res. 22 (25), 5600-5607 (1994).</li>
<li>Fellouse, F. A., Sidhu, S. S. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aFellouse%2C+FA+%22Making+and+Using+Antibodies%3A+A+Practical+Handbook%22">Making and Using Antibodies: A Practical Handbook</a>. , Second Edition, CRC Press. 157-180 (2006).</li>
<li>Fantini, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+antibody+library+diversity+through+next+generation+sequencing+and+technical+error+compensation%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Assessment of antibody library diversity through next generation sequencing and technical error compensation.</a> PLoS One. 12 (5), e0177574(2017).</li>
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</ol>

The authors have nothing to disclose.

To construct high diversity, phage-displayed Fab libraries, quality control check points are needed to monitor various stages of the construction process, including the competency of electro-competent cells, quality of the dU-ssDNA template, efficiency of CCC-dsDNA synthesis, titer after electroporation, Fab folding, and amino acid diversity of CDRs by sequence analysis of Fab-phage clones.
High yield and purity of dU-ssDNA is essential for high mutagenesis rate. In our experience, phage induc...

mAbs have broad applications ranging from basic research to disease diagnostics and therapeutics. As of 2016, more than 60 mAbs have been approved by the United States Food and Drug Administration (USFDA) for clinical treatment of autoimmune diseases, cancer, and infectious diseases1,2.
In 1975, Kohler and Milstein reported a technique for the continuous generation of antibodies of a single clonal specificity from a cellular source referred to as &#39;hybridomas&#39; and this technique has subsequently become a cornerstone in medicine and industry3,4. Generation of mAbs by this method requires various steps including antigen production, mouse immunization, extraction of B lymphocytes, fusion of B cells with myeloma cells to form immortal hybridoma cells, clone selection, and for therapeutic applications, humanization is required to avoid human anti-mouse antibody (HAMA)4,5. However, for this technology, antigens including toxins, pathogens, and highly conserved proteins are relatively ineffective in triggering an in vivo immune response for mAb production5.
In 1978, Hutchison et al. reported the use of an oligonucleotide to direct mutagenesis of a residue in a single-stranded bacteriophage virus6. In 1985, Smith reported that foreign gene fragments can be fused in frame with the gene encoding phage coat protein III and can thus be displayed on the phage surface without compromising its infectivity7. These pioneering works laid a foundation for the subsequent construction of phage-displayed antibody libraries in immune, na&#239;ve, and synthetic forms with the formats of single-chain variable fragment (scFv) and antigen-binding fragment (Fab) for therapeutic mAb development8,9. From the technical point of view, phage display-based antibody development offers a complementary approach to hybridoma-based mAb development that can help to circumvent the limitations some antigens can pose and the humanization process that hybridoma-derived antibodies often require5. As of 2016, 6 phage display-derived mAbs have been approved in the market including Humira, one of the most successful mAbs used for treatment of rheumatoid arthritis, and many phage display-derived antibody candidates are currently at various stages of clinical investigation10.
For immune and na&#239;ve phage antibody libraries, the diversity of complementarity-determining regions (CDRs) in light and heavy chain is derived from the natural immune repertoire (i.e., from B cells). In contrast, the diversity of CDRs in synthetic phage antibody libraries is entirely artificial. Synthetic approaches to library construction provide precise control over the design of sequence diversity and offer opportunities for mechanistic studies of antibody structure and function11,12. Moreover, the framework for synthetic libraries can be optimized before library construction to facilitate downstream, large-scale industrial development11,12.
In 1985, Kunkel reported a single-stranded DNA (ssDNA) template-based mutagenesis approach to introduce site-directed mutations into M13 bacteriophage efficiently13. This approach was subsequently used widely for construction of phage-displayed libraries. Chemically synthesized DNA oligonucleotides designed to introduce diversity into Fab CDRs are incorporated into a phagemid with an antibody backbone template. In this process, the phagemid is expressed as a uracil-containing ssDNA (dU-ssDNA) and the oligonucleotides are annealed onto the CDRs and extended to synthesize double-stranded DNA (dsDNA) in the presence of T7 DNA polymerase and T4 DNA ligase. Finally, generated ds-DNA can be introduced into Escherichia coli by electroporation.
For high diversity, phage-displayed library construction, high-voltage electroporation of a two-component mixture of electro-competent cells and covalently closed circular dsDNA (CCC-dsDNA) should be prepared carefully. Sidhu et al. modified the preparation of electro-competent cells and DNA from traditional methods and greatly improved library diversity14.
In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. Figure 1 shows an overview of library construction including: 1) high-efficiency electro-competent cell preparation; 2) extraction of dU-ssDNA; 3) Kunkel&#39;s method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based ELISA for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity. All the reagents, strains and equipment are listed in the Material&#39;s Table. Table 1 shows the reagent setup.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1.0 M H&lt;sub&gt;3&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>AC29570</td><td></td></tr><tr><td>1.0 M Tris, pH 8.0</td><td>Invitrogen</td><td>15568-025</td><td></td></tr><tr><td>10 mM ATP</td><td>Invitrogen</td><td>18330-019</td><td></td></tr><tr><td>100 mM dithiothreitol</td><td>Fisher</td><td>BP172</td><td></td></tr><tr><td>100 mM dNTP mix</td><td>GE Healthcare</td><td>28-4065-60</td><td>solution containing 25 mM each of dATP, dCTP, dGTP and dTTP.</td></tr><tr><td>3,3&amp;rsquo;,5,5&amp;rsquo;-tetramethylbenzidine (TMB)</td><td>Kirkegaard &amp;amp; Perry Laboratories Inc</td><td>50-76-02</td><td></td></tr><tr><td>50X TAE</td><td>Invitrogen</td><td>24710030</td><td></td></tr><tr><td>Agarose</td><td>Fisher</td><td>BP160</td><td></td></tr><tr><td>Carbenicillin, carb</td><td>Sigma</td><td>C1389</td><td>100 mg/mL in water,&amp;nbsp; 0.22 &amp;mu;m filter-sterilize, work concentration: 100 &amp;mu;g/mL.</td></tr><tr><td>Chloramphenicol, cmp</td><td>Sigma</td><td>C0378</td><td>100 mg/mL in ethanol, 0.22 &amp;mu;m filter-sterilize, work concentration: 10 &amp;mu;g/mL.</td></tr><tr><td>EDTA 0.5 M, pH 8.0</td><td>Invitrogen</td><td>AM9620G</td><td></td></tr><tr><td>Granulated agar</td><td>VWR</td><td>J637-500G</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; peroxidase substrate</td><td>Kirkegaard &amp;amp; Perry Laboratories Inc</td><td>50-65-02</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>795488</td><td></td></tr><tr><td>Kanamycin, kan</td><td>Fisher</td><td>AC61129</td><td>50 mg/mL in water,&amp;nbsp; 0.22 &amp;mu;m filter-sterilize, work concentration: 50 &amp;mu;g/mL.</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P2222</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>94046</td><td></td></tr><tr><td>NaCl</td><td>Alfa Aesar</td><td>U19C015</td><td></td></tr><tr><td>Nanodrop</td><td>Fisher</td><td>ND2000C</td><td></td></tr><tr><td>NaOH</td><td>Fisher</td><td>SS256</td><td>! CAUTION NaOH causes burns.</td></tr><tr><td>NON-Fat Powdered Milk</td><td>Sangon Biotech</td><td>A600669</td><td></td></tr><tr><td>PEG-8000</td><td>Fisher</td><td>BP233</td><td></td></tr><tr><td>Protein A-HRP conjugate</td><td>Invitrogen</td><td>101123</td><td></td></tr><tr><td>QIAprep Spin M13 Kit</td><td>Qiagen</td><td>22704</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28706</td><td></td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>Recombinant Protein L</td><td>Fisher</td><td>77679</td><td></td></tr><tr><td>T4 DNA polymerase</td><td>New England Biolabs</td><td>M0203S</td><td></td></tr><tr><td>T4 polynucleotide kinase</td><td>New England Biolabs</td><td>M0201S</td><td></td></tr><tr><td>T7 DNA polymerase</td><td>New England Biolabs</td><td>M0274S</td><td></td></tr><tr><td>Tetracycline, tet</td><td>Sigma</td><td>T7660</td><td>50 mg/mL in water, 0. 22 &amp;mu;m filter-sterilize, work concentration: 10 &amp;mu;g/mL.</td></tr><tr><td>Tryptone</td><td>Fisher</td><td>0123-07-5</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P2287</td><td></td></tr><tr><td>Ultrapure glycerol</td><td>Invitrogen</td><td>15514-011</td><td></td></tr><tr><td>Uridine</td><td>Sigma</td><td>U3750</td><td>25 mg/mL in ethanol, work concentration: 0.25 &amp;mu;g/mL.</td></tr><tr><td>Yeast extract</td><td>VWR</td><td>DF0127-08</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company &lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E.coli&lt;/em&gt; CJ236</td><td>New England Biolabs</td><td>E4141</td><td>Genotype: &lt;em&gt;dut&lt;sup&gt;-&lt;/sup&gt;&amp;nbsp; ung&lt;sup&gt;-&lt;/sup&gt;&amp;nbsp; thi-1 relA1 spoT1 mcrA/pCJ105&lt;/em&gt;(&lt;em&gt;F&#039; cam&lt;sup&gt;r&lt;/sup&gt;&lt;/em&gt;). Used for preparation of dU-ssDNA.</td></tr><tr><td>&lt;em&gt;E.coli&lt;/em&gt; SS320</td><td>Lucigen</td><td>60512</td><td>Genotype: [&lt;em&gt;F&#039;proAB+lacIq lacZ&amp;Delta;M15 Tn10 (tet&lt;sup&gt;r&lt;/sup&gt;)&lt;/em&gt;] &lt;em&gt;hsdR mcrB araD139 &amp;Delta;(araABC-leu)7679 &amp;Delta;lacX74 galUgalK rpsL thi&lt;/em&gt;. Optimized for high-efficiency electroporation and filamentous bacteriophage production.</td></tr><tr><td>M13KO7</td><td>New England Biolabs</td><td>N0315S</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company &lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.2-cm gap electroporation cuvette</td><td>BTX</td><td></td><td></td></tr><tr><td>96-well 2mL Deep-well plates</td><td>Fisher</td><td>278743</td><td></td></tr><tr><td>96-well Maxisorp immunoplates</td><td>Nunc</td><td>151759</td><td></td></tr><tr><td>Baffled flasks</td><td>Corning</td><td></td><td></td></tr><tr><td>Benchtop centrifuge</td><td>Eppendorf</td><td>5811000096</td><td></td></tr><tr><td>Centrifuge bottles</td><td>Nalgene</td><td></td><td></td></tr><tr><td>ECM-630 electroporator</td><td>BTX</td><td></td><td></td></tr><tr><td>Magnetic stir bars</td><td>Nalgene</td><td></td><td></td></tr><tr><td>Thermo Fisher centrifuge</td><td>Fisher</td><td></td><td></td></tr><tr><td>High speed shaker</td><td>TAITEK</td><td>MBR-034P</td><td></td></tr><tr><td>Microplate shaker</td><td>QILINBEIER</td><td>QB-9002</td><td></td></tr><tr><td>Liquid handler for 96 and 384 wells</td><td>RAININ</td><td></td><td></td></tr><tr><td>Mutil-channel pipette</td><td>RAININ</td><td>E4XLS</td><td></td></tr><tr><td>Amicon concentrator</td><td>Merck</td><td>UFC803096</td><td></td></tr></tbody></table>

construction of synthetic phage displayed fab library with tailored diversity

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel&#39;s method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.

Following the flow chart of the Fab library construction (see Figure 1), we prepared M13KO7 helper phage pre-infected E. coli SS320 electro-competent cells. The efficiency of these electro-competent cells is estimated as 2 X 109 cfu/µg when the Fab phagemid backbone for library construction was used (Figure 4).
The uracil incorporation efficiency by ...

This protocol describes a detailed procedure for the construction of a phage-displayed synthetic antibody library with tailored diversity. Synthetic antibodies have broad applications from basic research to disease diagnostics and therapeutics.

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb ...

construction-synthetic-phage-displayed-fab-library-with-tailored

Research

JoVE Journal

Immunology and Infection

In this video, we demonstrate the phage display technique to isolate pre-engineered phages expressing proteins with high affinity to the target protein.

Using Phage Display to Select Proteins with High Affinity to a Target Protein

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, ΦΧ174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

The protocols described allow the construction, characterization and selection (against the target of choice) of a "domainome" library made from any DNA source. This is achieved by a research pipeline that combines different technologies: phage display, a folding reporter and next generation sequencing with a web tool for data analysis.

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Ubiquitination is a critical protein post-translational modification, dysregulation of which has been implicated in numerous human diseases. This protocol details how phage display can be utilized to isolate novel ubiquitin variants that can bind and modulate the activity of E3 ligases that control the specificity, efficiency, and patterns of ubiquitination.

Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities.  Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers.  This method permits the isolation of all viral nucleic acid types from environmental samples.

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Learn to construct a synthetic phage-displayed human antibody library with tailored diversity for therapeutic monoclonal antibody development. This method bypasses animal immunization and enables high-affinity antibody selection.

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

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