Human ScFv Lib Generation

2011; doi: 10.1101/pdb.prot065573Cold Spring Harb Protoc IIIJennifer Andris-Widhopf, Peter Steinberger, Roberta Fuller, Christoph Rader and Carlos F. Barbas and Assembly of Light- and Heavy-Chain Coding SequencesGeneration of Human scFv Antibody Libraries: PCR Amplification

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Protocol

Generation of Human scFv Antibody Libraries: PCR Amplificationand Assembly of Light- and Heavy-Chain Coding Sequences

Jennifer Andris-Widhopf, Peter Steinberger, Roberta Fuller, Christoph Rader, and CarlosF. Barbas III

INTRODUCTION

The development of therapeutic antibodies for use in the treatment of human diseases has long been agoal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction ofhuman scFv (single chain antibody fragment) libraries using a short linker (GGSSRSS) or a long linker(GGSSRSSSSGGGGSGGGG). In this method, the individual rearranged heavy- and light-chain variableregions are amplified separately and are linked through a series of overlap polymerase chain reaction(PCR) steps to give the final scFv products that are used for cloning.

RELATED INFORMATION

Linkers of different compositions may result in different levels of oligomerization and may also alter theinterface of the VH and VL regions. Single-chain fragments in which the light- and heavy-chain variableregions are connected with a short peptide linker tend to form dimers, called bivalent diabodies,whereas scFvs with long linkers tend to be monomers (Holliger et al. 1993; McGuinness et al. 1996;Zhu et al. 1996). Bivalent diabodies can have the advantage of binding with higher avidity to theirantigen, but the use of a short linker can also lead to selection of unwanted low-affinity binders. Alterna-tive linkers that enhance scFv phage-binding activity have also been reported (Tang 1996; Turner et al.1997). The primers listed in this protocol are those used with the pComb3 vectors at the time ofthis writing.

A protocol describing the Generation of Human Fab Antibody Libraries: PCR Amplification andAssembly of Light- and Heavy-Chain Coding Sequences (Andris-Widhopf et al. 2011) is also available,as are protocols for Construction of cDNA Libraries Stage 1: Synthesis of First-Strand cDNACatalyzed by Reverse Transcriptase (Sambrook and Russell 2006a), Agarose Gel Electrophoresis(Sambrook and Russell 2006b), Standard Ethanol Precipitation of DNA in Microcentrifuge Tubes(Sambrook and Russell 2006c), Recovery of DNA from Agarose Gels: Electrophoresis onto DEAE-Cellulose Membranes (Sambrook and Russell 2006d), and Transformation of E. coli by Electropora-tion (Sambrook and Russell 2006e). Further information is also available on Quantitation of DNAand RNA (Barbas et al. 2007).

MATERIALS

RECIPES: Please see the end of this article for recipes indicated by <R>.

It is essential that you consult the appropriate Material Safety Data Sheets and your institution’sEnvironmental Health and Safety Office for proper handling of equipment and hazardous materialsused in this protocol.

Adapted from Phage Display: A Laboratory Manual (ed. Barbaset al.). CSHL Press, Cold Spring Harbor, NY, USA, 2001.Cite as: Cold Spring Harb Protoc; 2011; doi:10.1101/pdb.prot065573 www.cshprotocols.org

© 2011 Cold Spring Harbor Laboratory Press 1139

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Reagents

Agarose (Invitrogen)AmpliTaq DNA polymerase (Applied Biosystems)cDNA (see Construction of cDNA Libraries Stage 1: Synthesis of First-strand cDNA Catalyzed by

Reverse Transcriptase [Sambrook and Russell 2006a])DNA gel-loading dye (10×) <R>dNTP mix, 2.5 mM (dATP/dCTP/dGTP/dTTP set, 100 mM, GE Healthcare)E. coli, electrocompetent, e.g., XL1-Blue (Stratagene)Elutrap (Whatman)

A QIAEX II Gel Extraction Kit (QIAGEN) can be used as an alternative in Step 3.ii.LB agar + carbenicillin plates <R>MicroAmp PCR caps (Applied Biosystems)MicroAmp PCR trays (Applied Biosystems)MicroAmp PCR tubes (Applied Biosystems)Molecular weight marker, 100-bp DNA (GE Healthcare) or 1-kb DNA (Invitrogen)Oligonucleotide primers (human Fab; see Table 1)PCR buffer, 10× (supplied with Taq polymerase)Restriction enzyme buffer M, 10× (supplied with SfiI restriction enzyme)SfiI restriction enzyme, 40 units/µL (Roche Applied Science)<R>SOC medium for phage librariesT4 DNA ligase, 1 unit/µL (Invitrogen)T4 DNA ligase buffer, 5× (supplied with enzyme)TAE <R>

Dilute the 50× stock to prepare 1× TAE electrophoresis running buffer.Vector, pComb3HSS or pComb3XSS (available from the Barbas laboratory)

Equipment

Electroporation apparatus and materialsPCR cycler (e.g., GeneAmp 9700; Applied Biosystems)

Table 1. Human scFv primers, short and long linker

VH primers, 5′ sense, short linkerHSCVH1-F5′ GGT GGT TCC TCT AGA TCT TCC CAG GTG CAG CTG GTG CAG TCT GG 3′

HSCVH2-F5′ GGT GGT TCC TCT AGA TCT TCC CAG ATC ACC TTG AAG GAG TCT GG 3′

HSCVH35-F5′ GGT GGT TCC TCT AGA TCT TCC GAG GTG CAG CTG GTG SAG TCT GG 3′

HSCVH3a-F5′ GGT GGT TCC TCT AGA TCT TCC GAG GTG CAG CTG KTG GAG TCT G 3′

HSCVH4-F5′ GGT GGT TCC TCT AGA TCT TCC CAG GTG CAG CTG CAG GAG TCG GG 3′

HSCVH4a-F5′ GGT GGT TCC TCT AGA TCT TCC CAG GTG CAG CTA CAG CAG TGG GG 3′

VH primers, 5′ sense, long linkerHSCVH1-FL5′ GGT GGT TCC TCT AGA TCT TCC TCC TCT GGT GGC GGT GGC TCG GGC GGT GGT GGG CAG GTG CAG CTG GTG CAGTCT GG 3′

HSCVH2-FL5′ GGT GGT TCC TCT AGA TCT TCC TCC TCT GGT GGC GGT GGC TCG GGC GGT GGT GGG CAG ATC ACC TTG AAG GAGTCT GG 3′

HSCVH35-FL5′ GGT GGT TCC TCT AGA TCT TCC TCC TCT GGT GGC GGT GGC TCG GGC GGT GGT GGG GAG GTG CAG CTG GTG SAGTCT GG 3′

HSCVH3a-FL5′ GGT GGT TCC TCT AGA TCT TCC TCC TCT GGT GGC GGT GGC TCG GGC GGT GGT GGG GAG GTG CAG CTG KTG GAGTCT G 3′

HSCVH4-FL5′ GGT GGT TCC TCT AGA TCT TCC TCC TCT GGT GGC GGT GGC TCG GGC GGT GGT GGG CAG GTG CAG CTG CAG GAGTCG GG 3′

(continued)

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Table 1. Continued

HSCVH4a-FL5′ GGT GGT TCC TCT AGA TCT TCC TCC TCT GGT GGC GGT GGC TCG GGC GGT GGT GGG CAG GTG CAG CTA CAG CAGTGG GG 3′

VH primers, 3′ reverse, short and long linkerHSCG1234-B (corresponding to human IgG isotypes 1-4)5′ CCT GGC CGG CCT GGC CAC TAG TGA CCG ATG GGC CCT TGG TGG ARG C 3′

HSCM-B (corresponding to CH1 domain of human IgM, see Step 1.i)5′ CCT GGC CGG CCT GGC CAC TAG TAA GGG TTG GGG CGG ATG CAC TCC C 3′

HSCA-B (corresponding to CH1 domain of human IgA, see Step 1.i)5′ CCT GGC CGG CCT GGC CAC TAG TGA CCT TGG GGC TGG TCG GGG ATG C 3′

HSCD-B (corresponding to CH1 domain of human IgD, see Step 1.i)5′ CCT GGC CGG CCT GGC CAC TAG TCA CAT CCG GAG CCT TGG TGG GTG C 3′

HSCE-B (corresponding to CH1 domain of human IgE, see Step 1.i)5′ CCT GGC CGG CCT GGC CAC TAG TGA CGG ATG GGC TCT GTG TGG AGG C 3′

Vκ primers, 5′ sense, short and long linkerHSCK1-F5′ GGG CCC AGG CGG CCG AGC TCC AGA TGA CCC AGT CTC C 3′

HSCK24-F5′ GGG CCC AGG CGG CCG AGC TCG TGA TGA CYC AGT CTC C 3′

HSCK3-F5′ GGG CCC AGG CGG CCG AGC TCG TGW TGA CRC AGT CTC C 3′

HSCK5-F5′ GGG CCC AGG CGG CCG AGC TCA CAC TCA CGC AGT CTC C 3′

Vκ primers, 3′ reverse, short and long linkerHSCJK14o-B5′ GGA AGA TCT AGA GGA ACC ACC TTT GAT YTC CAC CTT GGT CCC 3′

HSCJK2o-B5′ GGA AGA TCT AGA GGA ACC ACC TTT GAT CTC CAG CTT GGT CCC 3′

HSCJK3o-B5′ GGA AGA TCT AGA GGA ACC ACC TTT GAT ATC CAC TTT GGT CCC 3′

HSCJK5o-B5′ GGA AGA TCT AGA GGA ACC ACC TTT AAT CTC CAG TCG TGT CCC 3′

Vλ primers, 5′ sense, short and long linkerHSCLam1a5′ GGG CCC AGG CGG CCG AGC TCG TGB TGA CGC AGC CGC CCT C 3′

HSCLam1b5′ GGG CCC AGG CGG CCG AGC TCG TGC TGA CTC AGC CAC CCT C 3′

HSCLam25′ GGG CCC AGG CGG CCG AGC TCG CCC TGA CTC AGC CTC CCT CCG T 3′

HSCLam35′ GGG CCC AGG CGG CCG AGC TCG AGC TGA CTC AGC CAC CCT CAG TGT C 3′

HSCLam45′ GGG CCC AGG CGG CCG AGC TCG TGC TGA CTC AAT CGC CCT C 3′

HSCLam65′ GGG CCC AGG CGG CCG AGC TCA TGC TGA CTC AGC CCC ACT C 3′

HSCLam785′ GGG CCC AGG CGG CCG AGC TCG TGG TGA CYC AGG AGC CMT C 3′

HSCLam95′ GGG CCC AGG CGG CCG AGC TCG TGC TGA CTC AGC CAC CTT C 3′

HSCLam105′ GGG CCC AGG CGG CCG AGC TCG GGC AGA CTC AGC AGC TCT C 3′

Vλ primers, 3′ reverse, short and long linkerHSCJLam12365′ GGA AGA TCT AGA GGA ACC ACC GCC TAG GAC GGT CAS CTT GGT SCC 3′

HSCJLam45′ GGA AGA TCT AGA GGA ACC ACC GCC TAA AAT GAT CAG CTG GGT TCC 3′

HSCJLam575′ GGA AGA TCT AGA GGA ACC ACC GCC GAG GAC GGT CAG CTS GGT SCC 3′

Overlap extension primersRSC-F (sense)5′ GAG GAG GAG GAG GAG GAG GCG GGG CCC AGG CGG CCG AGC TC 3′

RSC-B (reverse)5′ GAG GAG GAG GAG GAG GAG CCT GGC CGG CCT GGC CAC TAG TG 3′


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METHOD

Figure 1 shows a schematic overview of the steps that are used to generate the PCR products, and the flowchart in Figure 2depicts the entire library construction and selection procedure. This protocol describes the construction of κ and λ humanscFv libraries as separate entities. Depending on the needs of the user, the κ and λ light-chain products can be combinedprior to the overlap extension PCR such that only one kind of reaction is needed. Alternatively, the κ and λ scFv productscan be combined at a later step, before or after SfiI restriction digestion.

First Round PCR

PCR products and other DNA samples, precipitated or in solution, can be stored for years at −20˚C. The construction of anti-body fragment libraries, therefore, can be interrupted at any step.

A test amplification can be performed using the cDNA sample. If good amplification is not obtained (if a strong ampli-fied band is not clearly visible in the agarose gel), lithium chloride precipitation of the RNA might improve the results (seeConstruction of cDNA Libraries Stage 1: Synthesis of First-Strand cDNA Catalyzed by Reverse Transcriptase [Sambrookand Russell 2006a]).

1. To amplify the V gene rearrangements for a scFv with a short or long linker, perform 12 VH amplifica-tions, 16 Vκ amplifications, and 27 Vλ amplifications (see Figs. 3–5).

FIGURE 1. Generation of scFv fragments by PCR overlap extension for cloning into the pComb3 vector system. In the firstround of PCR, the rearranged light- and heavy-chain variable regions are amplified by using VL and VH sense primers in con-junction with JL and CH1 reverse primers (or JH reverse primers in chicken scFv libraries). The first-round products have a sizeof ~350–400 bp. The VL sense primers include a 5′ sequence tail that contains an SfiI site and is recognized by the senseextension primer. The CH1 (or JH) reverse primers introduce a 3′ sequence tail that contains an SfiI site and is recognized bythe reverse extension primer. The JL reverse and VH sense primers have overlapping sequence tails that code for the linkerpeptide in the final scFv PCR fragment. In the second-round PCR, the purified VL and VH products are fused by overlapextension PCR using the sense and reverse extension primers. The resulting product is ~750–800 bp in size and is referredto as the scFv PCR fragment. It has asymmetric SfiI restriction sites on the 5′ and 3′ ends that are used for directional cloninginto the pComb3 vectors.


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FIGURE 2. Flowchart depicting library construction and selection. Library construction begins with RNA preparation andcDNA synthesis, followed by preparation of PCR inserts for scFv (left) or Fab (right). During this preparation process,pComb3 vector is also prepared and tested for ligation efficiency. Prepared inserts are ligated into the prepared vectorand transformed into competent bacterial cells. Phage are rescued by the addition of helper phage and the panning/selec-tion process is started using the rescued phage. Panning is a cyclic procedure that is carried out in consecutive rounds overthe course of several days.

FIGURE 3. PCR amplification of human VH regions from cDNA.Each of the HSCVH-F (short linker) or HSCVH-FL (long linker)sense primers is paired with the reverse primers specific forthe 5′ end of the CH1 regions of the different immunoglobulinisotypes. In most cases, the use of the IgG- and IgM-specificprimers is sufficient. The sense primers have a sequence tailthat corresponds to the linker sequence used in the overlapextension PCR. Each reverse primer has a sequence tail con-taining an SfiI site; this tail is recognized by the reverse exten-sion primer used in the second-round PCR.


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i. Assemble one reaction containing the following reagents for each primer combination, usingspleen/bone marrow cDNA as template:

1 µL cDNA (0.5 µg)60 pmol 5′ primer (see primer combinations)60 pmol 3′ primer10 µL 10× PCR buffer8 µL 2.5 mM dNTPs0.5 µL Taq DNA polymerase

Add H2O to a final volume of 100 µL.VH primer combinations, short linker: VH primer combinations, long linker:

HSCVH1-F HSCVH2-F HSCVH1-FL HSCVH2-FLHSCG1234-B HSCG1234-B HSCG1234-B HSCG1234-B

HSCVH35-F HSCVH3a-F HSCVH35-FL HSCVH3a-FLHSCG1234-B HSCG1234-B HSCG1234-B HSCG1234-B

HSCVH4-F HSCVH4a-F HSCVH4-FL HSCVH4a-FLHSCG1234-B HSCG1234-B HSCG1234-B HSCG1234-B

HSCVH1-F HSCVH2-F HSCVH1-FL HSCVH2-FLHSCM-B HSCM-B HSCM-B HSCM-B

HSCVH35-F HSCVH3a-F HSCVH35-FL HSCVH3a-FLHSCM-B HSCM-B HSCM-B HSCM-B

HSCVH4-F HSCVH4a-F HSCVH4-FL HSCVH4a-FLHSCM-B HSCM-B HSCM-B HSCM-B

Vκ primer combinations, shortand long linker:

Vλ primer combinations, shortand long linker:

HSCK1-F HSCK24-F HSCLam1a HSCLam1bHSCJK14o-B HSCJK14o-B HSCJLam1236 HSCJLam1236

HSCK3-F HSCK5-F HSCLam2 HSCLam3HSCJK14o-B HSCJK14o-B HSCJLam1236 HSCJLam1236



FIGURE 4. The amplification of human Vκ sequences for the con-struction of scFv short- and long-linker libraries. Each of theHSCK-F sense primers is paired with each of the HSCJKo-Breverse primers to amplify human Vκ gene segments from cDNA.The sense primers have a 5′ sequence tail containing an SfiI site;this tail is recognized by the sense extension primer. The reverseprimers have a linker sequence tail that allows the overlap extensionwith the VH products in the second round of PCR.


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HSCK1-F HSCK24-F HSCLam10HSCJK3o-B HSCJK3o-B HSCJLam1236

HSCK3-F HSCK5-F HSCLam1a HSCLam1bHSCJK3o-B HSCJK3o-B HSCJLam4 HSCJLam4



HSCLam78 HSCLamHSCJLam4 HSCJLam4

HSCLam10HSCJLam4

HSCLam1a HSCLam1bHSCJLam57 HSCJLam57

HSCLam2 HSCLam3HSCJLam57 HSCJLam57

HSCLam4 HSCLam6HSCJLam57 HSCJLam57

HSCJLam78 HSCJLam9HSCJLam57 HSCJLam57

HSCJLam10HSCJLam57

Extra VH reverse primer sequences are provided in Table 1. These primers correspond to the CH1 domain of human IgA,IgD, and IgE. Traditionally, we make human libraries primarily from the IgM and IgG circulating pools of B lymphocytes, asthese are the isotypes that have the highest steady-state serum concentrations. The three additional isotypes may beprevalent in certain types of responses; e.g., IgE antibodies are elevated in allergic responses and IgA is most prevalentat mucosal sites. We recommend using these reverse primers to build libraries for such specific purposes. In each case,the VH sense primers listed below should be paired with the desired reverse primer as shown with IgM and IgG. Thesereverse primers can be used in the amplification of VH for both short linker and long linker scFv.

ii. Perform the PCR under the following conditions:

94˚C for 5 min30 cycles of 94˚C for 15 sec, 56˚C for 15 sec, 72˚C for 90 sec72˚C for 10 minA “hot start” PCR protocol can improve specificity, sensitivity, and yield. In hot start PCR, either an essential reac-tion component is not added until the first denaturing step, or a reversible inhibitor of the polymerase is used.This protocol prevents low-stringency primer extension, which can generate nonspecific products.

FIGURE 5. The amplification of human Vλ sequences for theconstruction of scFv short- and long-linker libraries. Each ofthe HSCLam sense primers is paired with each of theHSCJLam reverse primers to amplify human Vλ gene segmentsfrom cDNA. The sense primers have a 5′ sequence tail contain-ing an SfiI site; this tail is recognized by the sense extensionprimer. The reverse primers have a linker sequence tail thatallows the overlap extension with the VH products in thesecond round of PCR.


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2. Evaluate 5–10 µL of each reaction on a 2% agarose gel using DNA gel-loading dye and an appropriatemolecular weight marker (MWM).From these amplifications, expect a ~400-bp product for the VH reactions and a ~350-bp product for the Vκ and Vλreactions.

3. Isolate the PCR products:

i. Pool the products of each type of reaction, ethanol-precipitate, and wash as described inStandard Ethanol Precipitation of DNA in Microcentrifuge Tubes (Sambrook andRussell 2006c).

ii. Run the products on a 2% agarose gel, cut out the correct-sized bands, and purify the DNA asdescribed in Recovery of DNA from Agarose Gels: Electrophoresis onto DEAE-CelluloseMembranes (Sambrook and Russell 2006d), or by electroelution with an Elutrap, or resinbinding (e.g., QIAEX II Gel Extraction Kit).

iii. Quantitate yields by reading the optical density (O.D.) at 260 nm (1 O.D. unit = 50 µg/mL)(see Quantitation of DNA and RNA [Barbas et al. 2007]).Approximately 2–4 µg of each pool is required to proceed. If yields are too low, repeat the first round of PCR andcombine the end products.

Second Round PCR (Overlap Extension)

In the second round of PCR, the appropriate first-round products are mixed in equal ratios to generate the overlap product.The primers in the first-round PCR create complementary sequences in the downstream region of the light-chain variableregions and the upstream region of the heavy-chain variable regions that serve as the overlap for the extension of the full-length product (see Fig. 6).

4. Perform overlap extension PCR:

i. Assemble ten 100-µL reactions for each overlap using the following reagents:

100 ng appropriate first-round products (see template combinations)

60 pmol 5′ primer (RSC-F)60 pmol 3′ primer (RSC-B)10 µL 10× PCR buffer8 µL 2.5 mM dNTPs0.5 µL Taq DNA polymerase

Add H2O to a final volume of 100 µL.template combinations for scFv with a short linker:

100 ng short linker VH product 100 ng short linker VH product100 ng Vκ product 100 ng Vλ product

FIGURE 6. The second-round PCR to generate scFv PCR fragments. Equimolar quantities of light-chain variable and heavy-chain variable fragments are used to create the overlap extension product. The sense and reverse extension primers used inthe second-round PCR recognize the sequence tails that were generated in the first-round PCR.


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template combinations for scFv with a long linker:

100 ng long linker VH product 100 ng long linker VH product100 ng Vκ product 100 ng Vλ product

ii. Perform the second-round PCR under the following conditions:

94˚C for 5 min25 cycles of 94˚C for 15 sec, 56˚C for 15 sec, 72˚C for 2 min72˚C for 10 min

5. Evaluate 5–10 µL of each reaction on a 2% agarose gel using DNA gel-loading dye and an appropriateMWM for reference.For each type of reaction, expect a ~750- to 800-bp product. The long PCR extension time favors full-length product. If astrong band of the correct size is not clearly visible in the gel, the number of PCR cycles can be increased, or more overlapPCRs can be performed. To retain diversity of the library, it is better to increase the number of PCRs.

6. Isolate the PCR products as described in Step 3.i–3.iii.10–15 µg should be sufficient to continue. If yields are too low, repeat the overlap PCR and combine the end products.

Restriction Digestion

7. Prepare the overlap scFv PCR products and the pComb3HSS or pCOMB3XSS vector for cloning byperforming restriction digests with SfiI.:

i. Set up a digest of the PCR products containing the following reagents:

10 µg purified scFv product360 units SfiI (36 units per µg of DNA)20 µL 10× buffer M

Add water to a volume of 200 µL.

ii. Set up a digest of the vector containing the following reagents:

20 µg pComb3HSS or pComb3XSS (contains stuffer fragment between thetwo SfiI cloning sites)

120 units SfiI (6 units per µg of DNA)20 µL 10× buffer M

Add water to a volume of 200 µL.If the loss of digested PCR product in the final purification step is high or if several large-size ligations are neededto obtain a library of the desired size, digest more PCR product with SfiI. The amount of added enzyme shouldnot exceed 10% of the reaction volume. If desired, larger quantities of SfiI-cut and purified vector DNA can beprepared and tested in advance.

iii. Incubate both digests for 5 h at 50˚C.

8. Isolate the digested products:

i. (Optional) Ethanol-precipitate and wash as described in Standard Ethanol Precipitation ofDNA in Microcentrifuge Tubes (Sambrook and Russell 2006c).In some cases, SfiI-digested DNA is partly insoluble after ethanol precipitation. To avoid this problem, the DNAcan be loaded directly after digestion onto the agarose gel (Step 8.ii).

ii. Purify the digested scFv products on a 2% agarose gel, and purify the vector and stuffer frag-ment on a 1% agarose gel (see Agarose Gel Electrophoresis [Sambrook and Russell 2006b]).Let the DNA run long enough to separate linearized vector DNA (size ~5000 bp; cut only once)and uncut vector DNA from the desired double cut product (size ~3400 bp).We recommend electroelution with an Elutrap (Whatman) for extraction of the vector from the gel. Suboptimaldigestion or purification will yield vector with low transformation efficiencies and high background after ligation.


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The stuffer fragment can be used in a test ligation to determine the quality of the vector prior to use in libraryligations (Step 9.i). The stuffer fragment is ~1600 bp.

iii. Quantify the purified, digested PCR products, vector, and stuffer fragment by measuring theO.D. at 260 nm (see Quantitation of DNA and RNA [Barbas et al. 2007]).

Library Ligation

9. Perform small-scale ligations to assess the suitability of the vector and inserts for high-efficiency lig-ation and transformation.

i. Test the ligation efficiency of the vector DNA by ligating it with the gel-purified stuffer fragmentthat is generated during the SfiI digestion of the vector DNA, as follows:

Control ligation 1 (control insert):

140 ng pComb3HSS or pComb3XSS, SfiI-digested and purified140 ng stuffer fragment, SfiI-digested and purified4 µL 5× ligase buffer1 µL ligaseĆĆAdd H2O to a volume of 20 µL.

ii. Estimate the amount of uncut or partly cut vector DNA by setting up a ligation reaction thatcontains only vector DNA, as follows:

Control ligation 2 (test for vector self-ligation):

140 ng pComb3HSS or pComb3XSS, SfiI-digested and purified4 µL 5× ligase buffer1 µL ligaseĆĆAdd H2O to a volume of 20 µL.The vector preparation should contain little uncut vector DNA or DNA that is cut only once.

iii. Perform a small-scale test ligation in parallel with the two control ligations, and include thesame amount of vector DNA, as follows:

Small-scale test ligation (one reaction for each PCR insert):

140 ng pComb3HSS or pComb3XSS, SfiI-digested and purified70 ng scFv (short or long) overlap PCR product, SfiI-digested and purified4 µL 5× ligase buffer1 µL ligase

Add H2O to a volume of 20 µL.

iv. Incubate reactions between 4 h and overnight at room temperature.

10. Transform 0.5–1 µL of each reaction by electroporation into 50 µL of XL1-Blue electrocompetentcells. Dilute the transformed cultures 10-fold and 100-fold with prewarmed (37˚C) SOC, andplate 100 µL of each dilution on LB agar + carbenicillin plates.

11. Incubate the plates overnight at 37˚C. Count the colonies on the vector + Fab insert plates and cal-culate the number of transformants per µg pf vector DNA; if this number does not exceed 1 × 107, donot proceed with the large-scale library ligation. Determine the number of scaled-up library ligationsneeded to achieve the desired final library size.Ideally, the final library size should be several times 108 but at least 5 × 107 total transformants.

12. Count the colonies obtained fromControl ligation 1 and Control ligation 2 for an indication of vectorquality and ligation efficiency. If the background is >10% or the ligation efficiency is low, digest newvector and repeat the ligations. If the ligations are reasonably good, but there is not enough vector orinsert to make a library of the desired size, perform additional digests.


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A good vector DNA preparation should yield at least 108 colony-forming units (cfu) per µg of vector DNA and shouldhave <10% (ideally <5%) background ligation (calculated as cfu per µg of vector DNA in Control ligation 2).

13. Perform library ligation:i. Assemble enough reactions to produce at least 5 × 107 transformants. Combine the following:

1.4 µg pComb3HSS or pComb3XSS, SfiI-digested and purified700 ng scFv (short or long) overlap product, SfiI digested and purified40 µL 5× ligase buffer10 µL ligase

Add H2O to a volume of 200 µL.

ii. Incubate overnight at room temperature.

14. Ethanol-precipitate and wash the pellet as described in Standard Ethanol Precipitation of DNA inMicrocentrifuge Tubes (Sambrook and Russell 2006c).The ethanol-precipitated ligation reaction can be stored for months at −20˚C.

15. Transform the ligation reaction by resuspending in 15 µL of H2O and electroporating into XL1-Blueelectrocompetent cells, as described in Transformation of E. coli by Electroporation (Sambrookand Russell 2006e).

16. Proceed with the preparation and panning of the primary library.

REFERENCES

Andris-Widhopf J, Steinberger P, Fuller R, Rader C, Barbas CF III. 2011.Generation of human Fab antibody libraries: PCR amplification andassembly of light- and heavy-chain coding sequences. Cold SpringHarb Protoc doi: 10.1101/pdb.prot065565.

Barbas CF III, Burton DR, Scott JR, Silverman GJ. 2007. Quantitation ofDNA and RNA. Cold Spring Harb Protoc doi: 10.1101/pdb.ip47.

Holliger P, Prospero T, Winter G. 1993. “Diabodies”: Small bivalentand bispecific antibody fragments. Proc Natl Acad Sci 90:6444–6448.

McGuinness BT, Walter G, FitzGerald K, Shuler P, MahoneyW, DuncanAR, Hoogenboom HR. 1996. Phage diabody repertoires for selec-tion of large numbers of bi specific antibody fragments. Nat Bio-technol 14: 1149–1154.

Sambrook J, Russell DW. 2006a. Construction of cDNA libraries stage 1:Synthesis of first-strand cDNA catalyzed by reverse transcriptase.Cold Spring Harb Protoc doi: 10.1101/pdb.prot4065.

Sambrook J, Russell DW. 2006b. Agarose gel electrophoresis. ColdSpring Harb Protoc doi: 10.1101/pdb.prot4020.

Sambrook J, Russell DW. 2006c. Standard ethanol precipitation of DNAin microcentrifuge tubes. Cold Spring Harb Protoc doi: 10.1101/pdb.prot4456.

Sambrook J, Russell DW. 2006d. Recovery of DNA from agarose gels:Electrophoresis onto DEAE-cellulose membranes. Cold Spring HarbProtoc doi: 10.1101/pdb.prot3214.

Sambrook J, Russell DW. 2006e. Transformation of E. coli by electro-poration. Cold Spring Harb Protoc doi: 10.1101/pdb.prot3933.

Tang Y, Jiang N, Parakh C, Hilvert D. 1996. Selection of linkers for a cat-alytic single-chain antibody using phage display technology. J BiolChem 271: 15682–15686.

Turner DJ, Ritter MA, George AJT. 1997. Importance of the linker inexpression of single-chain Fv antibody fragments: Optimisationof peptide sequence using phage display technology. J ImmunolMethods 205: 43–54.

Zhu Z, Zapata G, Shalaby R, Snedecor B, Chen H, Carter P. 1996. Highlevel secretion of a humanized bispecific diabody from Escherichiacoli. Bio/Technology 14: 192–196.

RECIPES

Recipes for items marked with <R> are provided here. Additional recipes can be found online at http://www.cshprotocols.org/recipes.

DNA gel-loading dye (10X)

3.9 mL glycerol500 µL 10% (w/v) SDS200 µL 0.5 M EDTA0.025 g bromophenol blue0.025 g xylene cyanol

Bring to 10 mL total volume with H2O.


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LB agar+carbenicillin plates

LB agar (GIBCO/Invitrogen)Carbenicillin (50 mg/mL)

To prepare LB agar plates with 50 µg/mL carbenicillin, combine 32 g of LB agar with 1 L of H2O. Stir and autoclave for15 min at 121˚C. When cooled to 45˚C-50˚C, add 1 mL of carbenicillin stock solution (50 mg/mL). Pour into Petridishes and allow to solidify. Store at 4˚C.

SOC medium for phage libraries

20 g tryptone5 g yeast extract0.5 g NaCl186 mg KCl10 mL MgCl2 (1 M, sterile)20 mL glucose (1 M, filter-sterilized)

Bring first 4 ingredients to 1 L total volume with H2O, stir until dissolved, and titrate to pH 7. Sterilize by autoclavingon liquid cycle for 20 min at 121˚C at 15 psi. When cooled, add the last two ingredients. Using aseptic conditions,aliquot into 10-mL portions and store at room temperature.

TAE

Prepare a 50X stock solution in 1 L of H2O:242 g of Tris base57.1 mL of acetic acid (glacial)100 mL of 0.5 M EDTA (pH 8.0)

The 1X working solution is 40 mM Tris-acetate/1 mM EDTA.


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Documents

Human ScFv Lib Generation