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.

<ol><li>Singh, S., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/28799485">Monoclonal Antibodies: A Review.</a> Curr Clin Pharmacol. , (2017).</li>
<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>
<li>Kohler, G., Milstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Continuous+cultures+of+fused+cells+secreting+antibody+of+predefined+specificity%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Continuous cultures of fused cells secreting antibody of predefined specificity.</a> Nature. 256 (5517), 495-497 (1975).</li>
<li>Milstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+hybridoma+revolution%3A+an+offshoot+of+basic+research%5BTitle%5D)+AND+%22Bioessays%22%5BJournal%5D)">The hybridoma revolution: an offshoot of basic research.</a> Bioessays. 21 (11), 966-973 (1999).</li>
<li>Gray, A. C., Sidhu, S. S., Chandrasekera, P. C., Hendriksen, C. F., Borrebaeck, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal-Friendly+Affinity+Reagents%3A+Replacing+the+Needless+in+the+Haystack%5BTitle%5D)+AND+%22Trends+Biotechnol%22%5BJournal%5D)">Animal-Friendly Affinity Reagents: Replacing the Needless in the Haystack.</a> Trends Biotechnol. 34 (12), 960-969 (2016).</li>
<li>Hutchison, C. A. 3rd, et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutagenesis+at+a+specific+position+in+a+DNA+sequence%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Mutagenesis at a specific position in a DNA sequence.</a> J Biol Chem. 253 (18), 6551-6560 (1978).</li>
<li>Smith, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Filamentous+fusion+phage%3A+novel+expression+vectors+that+display+cloned+antigens+on+the+virion+surface%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface.</a> Science. 228 (4705), 1315-1317 (1985).</li>
<li>Sidhu, S. S. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSidhu%2C+SS+%22Phage+Display+in+Biotechnology+and+Drug+Discovery%22">Phage Display in Biotechnology and Drug Discovery</a>. , CRC Press. (2005).</li>
<li>Weiss, G. A., Watanabe, C. K., Zhong, A., Goddard, A., Sidhu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+mapping+of+protein+functional+epitopes+by+combinatorial+alanine+scanning%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Rapid mapping of protein functional epitopes by combinatorial alanine scanning.</a> Proc Natl Acad Sci U S A. 97 (16), 8950-8954 (2000).</li>
<li>Frenzel, A., Schirrmann, T., Hust, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phage+display-derived+human+antibodies+in+clinical+development+and+therapy%5BTitle%5D)+AND+%22MAbs%22%5BJournal%5D)">Phage display-derived human antibodies in clinical development and therapy.</a> MAbs. 8 (7), 1177-1194 (2016).</li>
<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>
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<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>
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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

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/&#181;g when the Fab phagemid backbone for library construction was used (Figure 4).
The uracil incorporation efficienc...

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Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

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 ...

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Research

JoVE Journal

Immunology and Infection

13.6K Views.  ShanghaiTech University. 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.

Video: Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

Biochemistry

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

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

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.

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

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

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

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

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.

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

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

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

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.

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

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

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

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.

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

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.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins with high specificity but low affinity. Through phage display, ubiquitin variants (UbVs) can be engineered such that they exhibit improved affinity over wildtype ubiquitin and maintain binding specificity to target proteins. Phage display utilizes a phagemid library, whereby the pIII coat protein of a filamentous M13 bacteriophage (chosen because it is displayed externally on the phage surface) is fused with UbVs. Specific residues of human wildtype ubiquitin are soft and randomized (i.e., there is a bias towards to native wildtype sequence) to generate UbVs so that deleterious changes in protein conformation are avoided while introducing the diversity necessary for promoting novel interactions with the target protein. During the phage display process, these UbVs are expressed and displayed on phage coat proteins and panned against a protein of interest. UbVs that exhibit favorable binding interactions with the target protein are retained, whereas poor binders are washed away and removed from the library pool. The retained UbVs, which are attached to the phage particle containing the UbV's corresponding phagemid, are eluted, amplified, and concentrated so that they can be panned against the same target protein in another round of phage display. Typically, up to five rounds of phage display are performed, during which a strong selection pressure is imposed against UbVs that bind weakly and/or promiscuously so that those with higher affinities are concentrated and enriched. Ultimately, UbVs that demonstrate higher specificity and/or affinity for the target protein than their wildtype counterparts are isolated and can be characterized through further experiments.

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.

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins ...

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

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

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.

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Biology

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

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 &#946;-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called &#34;domainome&#34;, will be well expressed and soluble, making them suitable for structural/functional studies.
By cloning and displaying the &#34;domainome&#34; directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification.
The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

The protocols described allow the construction, characterization and selection (against the target of choice) of a &#34;domainome&#34; 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.

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...

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

Source: Zhang, W., et al., <a href="https://app.jove.com/v/62950">Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases.</a> J. Vis. Exp. (2021)
In this video, we demonstrate the phage display technique to isolate pre-engineered phages expressing proteins with high affinity to the target protein.

Source: Zhang, W., et al., Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases. J. Vis. Exp. (2021)
In this video, we demonstrate ...

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Summary

Explore More Videos

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

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

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

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

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

Scalable High Throughput Selection From Phage-displayed Synthetic Antibody Libraries

Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases

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

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

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