Human SCFV Lib Generation

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Generation of Human scFv Antibody Libraries: PCR Amplification and Assembly of Light- and Heavy-Chain Coding Sequences
Jennifer Andris-Widhopf, Peter Steinberger, Roberta Fuller, Christoph Rader and Carlos F. Barbas III Cold Spring Harb Protoc 2011; doi: 10.1101/pdb.prot065573 Email Alerting Service Subject Categories
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Protocol
Generation of Human scFv Antibody Libraries: PCR Amplication and Assembly of Light- and Heavy-Chain Coding Sequences
Jennifer Andris-Widhopf, Peter Steinberger, Roberta Fuller, Christoph Rader, and Carlos F. Barbas III
INTRODUCTION
The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody eld. One way to obtain these antibodies is through phagedisplay libraries constructed from human lymphocytes. This protocol describes the construction of human 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 variable regions are amplied separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the nal scFv products that are used for cloning.
Cold Spring Harbor Protocols
RELATED INFORMATION
Linkers of different compositions may result in different levels of oligomerization and may also alter the interface of the VH and VL regions. Single-chain fragments in which the light- and heavy-chain variable regions 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 their antigen, but the use of a short linker can also lead to selection of unwanted low-afnity binders. Alternative 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 of this writing. A protocol describing the Generation of Human Fab Antibody Libraries: PCR Amplication and Assembly 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 cDNA Catalyzed 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 DEAECellulose Membranes (Sambrook and Russell 2006d), and Transformation of E. coli by Electroporation (Sambrook and Russell 2006e). Further information is also available on Quantitation of DNA and RNA (Barbas et al. 2007).
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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 institutions Environmental Health and Safety Ofce for proper handling of equipment and hazardous materials used in this protocol.
Adapted from Phage Display: A Laboratory Manual (ed. Barbas et al.). CSHL Press, Cold Spring Harbor, NY, USA, 2001. Cite as: Cold Spring Harb Protoc; 2011; doi:10.1101/pdb.prot065573 2011 Cold Spring Harbor Laboratory Press
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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 SI restriction enzyme) SI restriction enzyme, 40 units/L (Roche Applied Science) <R>SOC medium for phage libraries T4 DNA ligase, 1 unit/L (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 materials PCR cycler (e.g., GeneAmp 9700; Applied Biosystems)
Table 1. Human scFv primers, short and long linker
VH primers, 5 sense, short linker

HSCVH1-F 5 GGT GGT TCC TCT AGA TCT TCC CAG GTG CAG CTG GTG CAG TCT GG 3 HSCVH2-F 5 GGT GGT TCC TCT AGA TCT TCC CAG ATC ACC TTG AAG GAG TCT GG 3 HSCVH35-F 5 GGT GGT TCC TCT AGA TCT TCC GAG GTG CAG CTG GTG SAG TCT GG 3 HSCVH3a-F 5 GGT GGT TCC TCT AGA TCT TCC GAG GTG CAG CTG KTG GAG TCT G 3 HSCVH4-F 5 GGT GGT TCC TCT AGA TCT TCC CAG GTG CAG CTG CAG GAG TCG GG 3 HSCVH4a-F 5 GGT GGT TCC TCT AGA TCT TCC CAG GTG CAG CTA CAG CAG TGG GG 3
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VH primers, 5 sense, long linker

HSCVH1-FL 5 GGT GGT TCC TCT AGA TCT TCC TCC TCT GGT GGC GGT GGC TCG GGC GGT GGT GGG CAG GTG CAG CTG GTG CAG TCT GG 3 HSCVH2-FL 5 GGT GGT TCC TCT AGA TCT TCC TCC TCT GGT GGC GGT GGC TCG GGC GGT GGT GGG CAG ATC ACC TTG AAG GAG TCT GG 3 HSCVH35-FL 5 GGT GGT TCC TCT AGA TCT TCC TCC TCT GGT GGC GGT GGC TCG GGC GGT GGT GGG GAG GTG CAG CTG GTG SAG TCT GG 3 HSCVH3a-FL 5 GGT GGT TCC TCT AGA TCT TCC TCC TCT GGT GGC GGT GGC TCG GGC GGT GGT GGG GAG GTG CAG CTG KTG GAG TCT G 3 HSCVH4-FL 5 GGT GGT TCC TCT AGA TCT TCC TCC TCT GGT GGC GGT GGC TCG GGC GGT GGT GGG CAG GTG CAG CTG CAG GAG TCG GG 3
(continued)
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Table 1. Continued
HSCVH4a-FL 5 GGT GGT TCC TCT AGA TCT TCC TCC TCT GGT GGC GGT GGC TCG GGC GGT GGT GGG CAG GTG CAG CTA CAG CAG TGG GG 3
VH primers, 3 reverse, short and long linker

HSCG1234-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
V primers, 5 sense, short and long linker

HSCK1-F 5 GGG CCC AGG CGG CCG AGC TCC AGA TGA CCC AGT CTC C 3 HSCK24-F 5 GGG CCC AGG CGG CCG AGC TCG TGA TGA CYC AGT CTC C 3 HSCK3-F 5 GGG CCC AGG CGG CCG AGC TCG TGW TGA CRC AGT CTC C 3 HSCK5-F 5 GGG CCC AGG CGG CCG AGC TCA CAC TCA CGC AGT CTC C 3
V primers, 3 reverse, short and long linker

HSCJK14o-B 5 GGA AGA TCT AGA GGA ACC ACC TTT GAT YTC CAC CTT GGT CCC 3 HSCJK2o-B 5 GGA AGA TCT AGA GGA ACC ACC TTT GAT CTC CAG CTT GGT CCC 3 HSCJK3o-B 5 GGA AGA TCT AGA GGA ACC ACC TTT GAT ATC CAC TTT GGT CCC 3 HSCJK5o-B 5 GGA AGA TCT AGA GGA ACC ACC TTT AAT CTC CAG TCG TGT CCC 3
V primers, 5 sense, short and long linker

HSCLam1a 5 GGG CCC AGG CGG CCG AGC TCG TGB TGA CGC AGC CGC CCT C 3 HSCLam1b 5 GGG CCC AGG CGG CCG AGC TCG TGC TGA CTC AGC CAC CCT C 3 HSCLam2 5 GGG CCC AGG CGG CCG AGC TCG CCC TGA CTC AGC CTC CCT CCG T 3 HSCLam3 5 GGG CCC AGG CGG CCG AGC TCG AGC TGA CTC AGC CAC CCT CAG TGT C 3 HSCLam4 5 GGG CCC AGG CGG CCG AGC TCG TGC TGA CTC AAT CGC CCT C 3 HSCLam6 5 GGG CCC AGG CGG CCG AGC TCA TGC TGA CTC AGC CCC ACT C 3 HSCLam78 5 GGG CCC AGG CGG CCG AGC TCG TGG TGA CYC AGG AGC CMT C 3 HSCLam9 5 GGG CCC AGG CGG CCG AGC TCG TGC TGA CTC AGC CAC CTT C 3 HSCLam10 5 GGG CCC AGG CGG CCG AGC TCG GGC AGA CTC AGC AGC TCT C 3
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V primers, 3 reverse, short and long linker

HSCJLam1236 5 GGA AGA TCT AGA GGA ACC ACC GCC TAG GAC GGT CAS CTT GGT SCC 3 HSCJLam4 5 GGA AGA TCT AGA GGA ACC ACC GCC TAA AAT GAT CAG CTG GGT TCC 3 HSCJLam57 5 GGA AGA TCT AGA GGA ACC ACC GCC GAG GAC GGT CAG CTS GGT SCC 3
Overlap extension primers

RSC-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 owchart in Figure 2 depicts the entire library construction and selection procedure. This protocol describes the construction of and human scFv libraries as separate entities. Depending on the needs of the user, the and light-chain products can be combined prior to the overlap extension PCR such that only one kind of reaction is needed. Alternatively, the and scFv products can be combined at a later step, before or after SI restriction digestion.
First Round PCR

PCR products and other DNA samples, precipitated or in solution, can be stored for years at 20C. The construction of antibody fragment libraries, therefore, can be interrupted at any step. A test amplication can be performed using the cDNA sample. If good amplication is not obtained (if a strong amplied band is not clearly visible in the agarose gel), lithium chloride precipitation of the RNA might improve the results (see Construction of cDNA Libraries Stage 1: Synthesis of First-Strand cDNA Catalyzed by Reverse Transcriptase [Sambrook and Russell 2006a]). 1. To amplify the V gene rearrangements for a scFv with a short or long linker, perform 12 VH amplica-
tions, 16 V amplications, and 27 V amplications (see Figs. 35).
PROTOCOLS
FIGURE 1. Generation of scFv fragments by PCR overlap extension for cloning into the pComb3 vector system. In the first round of PCR, the rearranged light- and heavy-chain variable regions are amplified by using VL and VH sense primers in conjunction with JL and CH1 reverse primers (or JH reverse primers in chicken scFv libraries). The first-round products have a size of ~350400 bp. The VL sense primers include a 5 sequence tail that contains an SfiI site and is recognized by the sense extension primer. The CH1 (or JH) reverse primers introduce a 3 sequence tail that contains an SfiI site and is recognized by the reverse extension primer. The JL reverse and VH sense primers have overlapping sequence tails that code for the linker peptide in the final scFv PCR fragment. In the second-round PCR, the purified VL and VH products are fused by overlap extension PCR using the sense and reverse extension primers. The resulting product is ~750800 bp in size and is referred to as the scFv PCR fragment. It has asymmetric SfiI restriction sites on the 5 and 3 ends that are used for directional cloning into the pComb3 vectors.
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FIGURE 2. Flowchart depicting library construction and selection. Library construction begins with RNA preparation and cDNA 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 vector and transformed into competent bacterial cells. Phage are rescued by the addition of helper phage and the panning/selection process is started using the rescued phage. Panning is a cyclic procedure that is carried out in consecutive rounds over the course of several days.
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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 for the 5 end of the CH1 regions of the different immunoglobulin isotypes. In most cases, the use of the IgG- and IgM-specific primers is sufficient. The sense primers have a sequence tail that corresponds to the linker sequence used in the overlap extension PCR. Each reverse primer has a sequence tail containing an SfiI site; this tail is recognized by the reverse extension primer used in the second-round PCR.
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FIGURE 4. The amplification of human V sequences for the construction of scFv short- and long-linker libraries. Each of the HSCK-F sense primers is paired with each of the HSCJKo-B reverse primers to amplify human V 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 reverse primers have a linker sequence tail that allows the overlap extension with the VH products in the second round of PCR.
i. Assemble one reaction containing the following reagents for each primer combination, using
spleen/bone marrow cDNA as template: 1 L 60 pmol 60 pmol 10 L 8 L 0.5 L cDNA (0.5 g) 5 primer (see primer combinations) 3 primer 10 PCR buffer 2.5 mM dNTPs Taq DNA polymerase
Add H2O to a nal volume of 100 L. VH primer combinations, short linker: HSCVH1-F HSCG1234-B
VH primer combinations, long linker: HSCVH1-FL HSCG1234-B HSCVH35-FL HSCG1234-B HSCVH4-FL HSCG1234-B HSCVH1-FL HSCM-B HSCVH35-FL HSCM-B HSCVH4-FL HSCM-B HSCVH2-FL HSCG1234-B HSCVH3a-FL HSCG1234-B HSCVH4a-FL HSCG1234-B HSCVH2-FL HSCM-B HSCVH3a-FL HSCM-B HSCVH4a-FL HSCM-B
HSCVH2-F HSCG1234-B HSCVH3a-F HSCG1234-B HSCVH4a-F HSCG1234-B HSCVH2-F HSCM-B HSCVH3a-F HSCM-B HSCVH4a-F HSCM-B
HSCVH35-F HSCG1234-B HSCVH4-F HSCG1234-B HSCVH1-F HSCM-B HSCVH35-F HSCM-B HSCVH4-F HSCM-B
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V primer combinations, short and long linker: HSCK1-F HSCJK14o-B HSCK3-F HSCJK14o-B HSCK1-F HSCJK2o-B HSCK3-F HSCJK2o-B HSCK24-F HSCJK14o-B HSCK5-F HSCJK14o-B HSCK24-F HSCJK2o-B HSCK5-F HSCJK2o-B
V primer combinations, short and long linker: HSCLam1a HSCJLam1236 HSCLam2 HSCJLam1236 HSCLam4 HSCJLam1236 HSCLam78 HSCJLam1236 HSCLam1b HSCJLam1236 HSCLam3 HSCJLam1236 HSCLam6 HSCJLam1236 HSCLam9 HSCJLam1236
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FIGURE 5. The amplification of human V sequences for the construction of scFv short- and long-linker libraries. Each of the HSCLam sense primers is paired with each of the HSCJLam reverse primers to amplify human V 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 reverse primers have a linker sequence tail that allows the overlap extension with the VH products in the second round of PCR.
HSCK1-F HSCJK3o-B HSCK3-F HSCJK3o-B HSCK1-F HSCJK5o-B HSCK3-F HSCJK5o-B
HSCK24-F HSCJK3o-B HSCK5-F HSCJK3o-B HSCK24-F HSCJK5o-B HSCK5-F HSCJK5o-B
HSCLam10 HSCJLam1236 HSCLam1a HSCJLam4 HSCLam2 HSCJLam4 HSCLam4 HSCJLam4 HSCLam78 HSCJLam4 HSCLam10 HSCJLam4 HSCLam1a HSCJLam57 HSCLam2 HSCJLam57 HSCLam4 HSCJLam57 HSCJLam78 HSCJLam57 HSCJLam10 HSCJLam57 HSCLam1b HSCJLam57 HSCLam3 HSCJLam57 HSCLam6 HSCJLam57 HSCJLam9 HSCJLam57 HSCLam1b HSCJLam4 HSCLam3 HSCJLam4 HSCLam6 HSCJLam4 HSCLam HSCJLam4
PROTOCOLS
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, as these are the isotypes that have the highest steady-state serum concentrations. The three additional isotypes may be prevalent in certain types of responses; e.g., IgE antibodies are elevated in allergic responses and IgA is most prevalent at mucosal sites. We recommend using these reverse primers to build libraries for such specic purposes. In each case, the VH sense primers listed below should be paired with the desired reverse primer as shown with IgM and IgG. These reverse primers can be used in the amplication of VH for both short linker and long linker scFv. ii. Perform the PCR under the following conditions:
94C for 5 min 30 cycles of 94C for 15 sec, 56C for 15 sec, 72C for 90 sec 72C for 10 min
A hot start PCR protocol can improve specicity, sensitivity, and yield. In hot start PCR, either an essential reaction component is not added until the rst denaturing step, or a reversible inhibitor of the polymerase is used. This protocol prevents low-stringency primer extension, which can generate nonspecic products.
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2. Evaluate 510 L of each reaction on a 2% agarose gel using DNA gel-loading dye and an appropriate
molecular weight marker (MWM).

From these amplications, expect a ~400-bp product for the VH reactions and a ~350-bp product for the V and V reactions. 3. Isolate the PCR products:
i. Pool the products of each type of reaction, ethanol-precipitate, and wash as described in Standard Ethanol Precipitation of DNA in Microcentrifuge Tubes (Sambrook and Russell 2006c). ii. Run the products on a 2% agarose gel, cut out the correct-sized bands, and purify the DNA as described in Recovery of DNA from Agarose Gels: Electrophoresis onto DEAE-Cellulose Membranes (Sambrook and Russell 2006d), or by electroelution with an Elutrap, or resin binding (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 g/mL) (see Quantitation of DNA and RNA [Barbas et al. 2007]).
Approximately 24 g of each pool is required to proceed. If yields are too low, repeat the rst round of PCR and combine the end products.
Second Round PCR (Overlap Extension)

In the second round of PCR, the appropriate rst-round products are mixed in equal ratios to generate the overlap product. The primers in the rst-round PCR create complementary sequences in the downstream region of the light-chain variable regions and the upstream region of the heavy-chain variable regions that serve as the overlap for the extension of the fulllength product (see Fig. 6). 4. Perform overlap extension PCR: i. Assemble ten 100-L reactions for each overlap using the following reagents:
100 ng appropriate rst-round products (see template combinations) 60 pmol 60 pmol 10 L 8 L 0.5 L 5 primer (RSC-F) 3 primer (RSC-B) 10 PCR buffer 2.5 mM dNTPs Taq DNA polymerase
PROTOCOLS
Add H2O to a nal volume of 100 L. template combinations for scFv with a short linker: 100 ng short linker VH product 100 ng V product 100 ng short linker VH product 100 ng V product
FIGURE 6. The second-round PCR to generate scFv PCR fragments. Equimolar quantities of light-chain variable and heavychain variable fragments are used to create the overlap extension product. The sense and reverse extension primers used in the 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 V product 100 ng long linker VH product 100 ng V product
ii. Perform the second-round PCR under the following conditions:
94C for 5 min 25 cycles of 94C for 15 sec, 56C for 15 sec, 72C for 2 min 72C for 10 min
5. Evaluate 510 L of each reaction on a 2% agarose gel using DNA gel-loading dye and an appropriate
MWM for reference.

For each type of reaction, expect a ~750- to 800-bp product. The long PCR extension time favors full-length product. If a strong band of the correct size is not clearly visible in the gel, the number of PCR cycles can be increased, or more overlap PCRs 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.i3.iii. 1015 g should be sufcient 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 by
performing restriction digests with SI.:

i. Set up a digest of the PCR products containing the following reagents:
10 g 360 units 20 L
puried scFv product SI (36 units per g of DNA) 10 buffer M
Add water to a volume of 200 L.

ii. Set up a digest of the vector containing the following reagents:
20 g 120 units 20 L 10 buffer M
pComb3HSS or pComb3XSS (contains stuffer fragment between the two SI cloning sites) SI (6 units per g of DNA)
PROTOCOLS
Add water to a volume of 200 L.

If the loss of digested PCR product in the nal purication step is high or if several large-size ligations are needed to obtain a library of the desired size, digest more PCR product with SI. The amount of added enzyme should not exceed 10% of the reaction volume. If desired, larger quantities of SI-cut and puried vector DNA can be prepared 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 of
DNA in Microcentrifuge Tubes (Sambrook and Russell 2006c).

In some cases, SI-digested DNA is partly insoluble after ethanol precipitation. To avoid this problem, the DNA can 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. Suboptimal digestion or purication will yield vector with low transformation efciencies 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 library ligations (Step 9.i). The stuffer fragment is ~1600 bp. iii. Quantify the puried, digested PCR products, vector, and stuffer fragment by measuring the
O.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-efciency lig-
ation and transformation.

i. Test the ligation efciency of the vector DNA by ligating it with the gel-puried stuffer fragment
that is generated during the SI digestion of the vector DNA, as follows: Control ligation 1 (control insert): 140 ng 140 ng 4 L 1 L pComb3HSS or pComb3XSS, SI-digested and puried stuffer fragment, SI-digested and puried 5 ligase buffer ligase
Add H2O to a volume of 20 L.

ii. Estimate the amount of uncut or partly cut vector DNA by setting up a ligation reaction that
contains only vector DNA, as follows: Control ligation 2 (test for vector self-ligation): 140 ng 4 L 1 L pComb3HSS or pComb3XSS, SI-digested and puried 5 ligase buffer ligase
Add H2O to a volume of 20 L. 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 the
same amount of vector DNA, as follows: Small-scale test ligation (one reaction for each PCR insert): 140 ng 70 ng 4 L 1 L pComb3HSS or pComb3XSS, SI-digested and puried scFv (short or long) overlap PCR product, SI-digested and puried 5 ligase buffer ligase
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iv. Incubate reactions between 4 h and overnight at room temperature. 10. Transform 0.51 L of each reaction by electroporation into 50 L of XL1-Blue electrocompetent
cells. Dilute the transformed cultures 10-fold and 100-fold with prewarmed (37C) SOC, and plate 100 L 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 g pf vector DNA; if this number does not exceed 1 107, do not proceed with the large-scale library ligation. Determine the number of scaled-up library ligations needed to achieve the desired nal library size.
Ideally, the nal library size should be several times 108 but at least 5 107 total transformants. 12. Count the colonies obtained from Control ligation 1 and Control ligation 2 for an indication of vector
quality and ligation efciency. If the background is >10% or the ligation efciency is low, digest new vector and repeat the ligations. If the ligations are reasonably good, but there is not enough vector or insert 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 g of vector DNA and should have <10% (ideally <5%) background ligation (calculated as cfu per g of vector DNA in Control ligation 2). 13. Perform library ligation: 7 i. Assemble enough reactions to produce at least 5 10 transformants. Combine the following:
1.4 g 700 ng 40 L 10 L
pComb3HSS or pComb3XSS, SI-digested and puried scFv (short or long) overlap product, SI digested and puried 5 ligase buffer ligase

ii. Incubate overnight at room temperature. 14. Ethanol-precipitate and wash the pellet as described in Standard Ethanol Precipitation of DNA in
Microcentrifuge Tubes (Sambrook and Russell 2006c).

The ethanol-precipitated ligation reaction can be stored for months at 20C. 15. Transform the ligation reaction by resuspending in 15 L of H2O and electroporating into XL1-Blue
electrocompetent cells, as described in Transformation of E. coli by Electroporation (Sambrook and 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 amplication and assembly of light- and heavy-chain coding sequences. Cold Spring Harb Protoc doi: 10.1101/pdb.prot065565. Barbas CF III, Burton DR, Scott JR, Silverman GJ. 2007. Quantitation of DNA and RNA. Cold Spring Harb Protoc doi: 10.1101/pdb.ip47. Holliger P, Prospero T, Winter G. 1993. Diabodies: Small bivalent and bispecic antibody fragments. Proc Natl Acad Sci 90: 64446448. McGuinness BT, Walter G, FitzGerald K, Shuler P, Mahoney W, Duncan AR, Hoogenboom HR. 1996. Phage diabody repertoires for selection of large numbers of bi specic antibody fragments. Nat Biotechnol 14: 11491154. Sambrook J, Russell DW. 2006a. Construction of cDNA libraries stage 1: Synthesis of rst-strand cDNA catalyzed by reverse transcriptase. Cold Spring Harb Protoc doi: 10.1101/pdb.prot4065. Sambrook J, Russell DW. 2006b. Agarose gel electrophoresis. Cold Spring Harb Protoc doi: 10.1101/pdb.prot4020. Sambrook J, Russell DW. 2006c. Standard ethanol precipitation of DNA in 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 Harb Protoc doi: 10.1101/pdb.prot3214. Sambrook J, Russell DW. 2006e. Transformation of E. coli by electroporation. Cold Spring Harb Protoc doi: 10.1101/pdb.prot3933. Tang Y, Jiang N, Parakh C, Hilvert D. 1996. Selection of linkers for a catalytic single-chain antibody using phage display technology. J Biol Chem 271: 1568215686. Turner DJ, Ritter MA, George AJT. 1997. Importance of the linker in expression of single-chain Fv antibody fragments: Optimisation of peptide sequence using phage display technology. J Immunol Methods 205: 4354. Zhu Z, Zapata G, Shalaby R, Snedecor B, Chen H, Carter P. 1996. High level secretion of a humanized bispecic diabody from Escherichia coli. Bio/Technology 14: 192196.
PROTOCOLS
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 glycerol 500 L 10% (w/v) SDS 200 L 0.5 M EDTA 0.025 g bromophenol blue 0.025 g xylene cyanol Bring to 10 mL total volume with H2O.
1149
LB agar+carbenicillin plates
LB agar (GIBCO/Invitrogen) Carbenicillin (50 mg/mL) To prepare LB agar plates with 50 g/mL carbenicillin, combine 32 g of LB agar with 1 L of H2O. Stir and autoclave for 15 min at 121C. When cooled to 45C-50C, add 1 mL of carbenicillin stock solution (50 mg/mL). Pour into Petri dishes and allow to solidify. Store at 4C.
SOC medium for phage libraries

20 g tryptone 5 g yeast extract 0.5 g NaCl 186 mg KCl 10 mL MgCl2 (1 M, sterile) 20 mL glucose (1 M, lter-sterilized) Bring rst 4 ingredients to 1 L total volume with H2O, stir until dissolved, and titrate to pH 7. Sterilize by autoclaving on liquid cycle for 20 min at 121C 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 base 57.1 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.
PROTOCOLS
1150

Human SCFV Lib Generation

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2011 Cold Spring Harbor Laboratory Press

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Dilute the 50 stock to prepare 1 TAE electrophoresis running buffer.

Vector, pComb3HSS or pComb3XSS (available from the Barbas laboratory)

Table 1. Human scFv primers, short and long linker

VH primers, 5 sense, short linker

VH primers, 5 sense, long linker

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VH primers, 3 reverse, short and long linker

V primers, 5 sense, short and long linker

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V primers, 3 reverse, short and long linker

V primers, 5 sense, short and long linker

V primers, 3 reverse, short and long linker

Overlap extension primers

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First Round PCR

tions, 16 V amplications, and 27 V amplications (see Figs. 35).

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Cold Spring Harbor Protocols

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HSCK1-F HSCJK3o-B HSCK3-F HSCJK3o-B HSCK1-F HSCJK5o-B HSCK3-F HSCJK5o-B

HSCK24-F HSCJK3o-B HSCK5-F HSCJK3o-B HSCK24-F HSCJK5o-B HSCK5-F HSCJK5o-B

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Cold Spring Harbor Protocols

molecular weight marker (MWM).

Second Round PCR (Overlap Extension)

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Cold Spring Harbor Protocols

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

MWM for reference.

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performing restriction digests with SI.:

puried scFv product SI (36 units per g of DNA) 10 buffer M

Add water to a volume of 200 L.

20 g 120 units 20 L 10 buffer M

Add water to a volume of 200 L.

DNA in Microcentrifuge Tubes (Sambrook and Russell 2006c).

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ation and transformation.

Add H2O to a volume of 20 L.

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Add H2O to a volume of 20 L.

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Add H2O to a volume of 200 L.

Microcentrifuge Tubes (Sambrook and Russell 2006c).

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DNA gel-loading dye (10X)

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SOC medium for phage libraries

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Cold Spring Harbor Protocols

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