Antibody Phage Display Methods and Protocols - part 10 pps

These immunoglobulin G molecules are composed of heavy-chain dimers and are devoid of light chains. Furthermore, these molecules lack the C H 1 domain. Expression of the binding domain (V HH ) of these heavy chain Abs in S. cerevisiae resulted in the effi cient secretion of this molecule and production levels of 250 mg/L were obtained in shake-fl ask experiments (4). We have shown that the level of V HH expression is dramatically higher than that seen when the same Ab fragment is expressed in Escherichia coli (Fig. 1). This chapter describes protocols for the expression of heavy chain Ab fragments (V HH ) in S. cerevisiae using an episomal yeast expression plasmid (pUR4548) under control of the GAL7 promoter. The recombinant plasmid is transformed into yeast by lithium acetate transformation or electroporation and the expressed Ab fragments are extracted from the yeast growth medium. More detailed information on yeast expression systems can be found in Romanos et al. (5). 2. Materials 1. Yeast strain for expression. A typical yeast strain used in this laboratory is SU51 (can1, his4, 519, leu2, 3, 112, cir + ; 6). This strain grows in the presence of histidine, leucine, and a proper carbon source (see Subheading 2.7. for selective minimal medium, and Subheading 2.8. for rich, nonselective medium for this strain). 2. Plasmid vector suitable for Ab expression in yeast. The plasmid used in the laboratory, pUR4548 (4), contains the LEU2 selection marker, the SUC2 signal sequence for protein secretion, and the galactose-inducible GAL7 promoter (see Notes 1–2). 3. Distilled H 2 O used for the preparation of buffers and growth media should be double-autoclaved. Used glassware should be free of any contaminants (an overnight incubation with 100 mM HCl, followed by washing and autoclaving, is suffi cient). 4. SD broth: 10X stock solution: 6.7 g yeast nitrogen base (without amino acids) dissolved in 100 mL H 2 O; fi lter-sterilize through a 0.22 µmfi lter. 5. 20% Glucose 10X stock solution, sterilized by autoclaving (20 min at 120°C/ 1.4 bar). 6. Amino acid stock solutions: 100X stocks are prepared in distilled water then fi lter-sterilized through 0.22 µmfi lters. Final concentrations for the most com- monly used amino acids are: L-adenine, 0.4 mg/mL; L-valine, 1.5 mg/mL; L-histidine, 0.2 mg/mL; uracil, 0.2 mg/mL; L-leucine, 0.6 mg/mL. 7. YEPD broth: 1% (w/v) yeast extract and 2% (w/v) peptone. Autoclave, then add 2% (v/v) glucose. 8. 1.0 M LiAC: 1.02 g in 10 mL distilled water; fi lter-sterilize through a 0.22 µm fi lter. 9. 50% (w/v) Polyethylene glycol (PEG) 4000 in distilled water. Autoclave. 360 van der Vaart 10. TE buffer: 10 mM Tris-HCl, pH 7.5, 1 mM ethylene diamine tetraacetic acid, pH 8.0. Autoclave. 11. Salmon testes carrier DNA (Sigma D1626): 2 mg/mL TE buffer. Mix vigorously on a magnetic stirrer for 2–3 h or until fully dissolved. If convenient, leave the covered solution mixing overnight in a cold room. Aliquot the DNA into 1 mL vol and store at –20°C. Before use, boil for 5 min and chill quickly on ice (see Note 3). 12. SD agar (for use if an auxotrophic marker is complemented in the yeast strain): Dissolve 15 g Bacto-agar in 790 mL distilled water and autoclave. Cool to 60°C and add 100 mL 10X stock of yeast nitrogen base without amino acids, 100 mL 10X glucose stock, and 10 mL 100X stock(s) of amino acids as appropri- ate (see above). 13. YEPD agar (for use if a dominant marker has been introduced into the yeast strain): 1.5% (w/v) Bacto-agar, 1% (w/v) yeast extract, and 2% (w/v) peptone. Autoclave. Depending on the dominant selection marker used, antibiotics and carbon sources may need to be added. Fig. 1. Comparison of llama Ab fragment (V HH ) production in E. coli and S. cerevisiae. The periplasmic fraction of E. coli and growth medium of an S. cerevisiae strain expressing an identical llama Ab fragment were separated on a 14% SDS-PAGE gel. After separation, the protein bands were visualized by Coomassie blue staining. The molecular weight of the V HH protein band is approx 13 kDa. Expression of V HH Ab Fragments 361 14. HEPES–dithiothreitol buffer: 20 mM HEPES, 25 mM dithiothreitol in YEP containing 2% (w/v) glucose. Filter-sterilize through a 0.22-µmfi lter. Prepare freshly before use. 15. 1 M Sorbitol. Autoclave. 16. Electroporation cuvets, 2 mm gap, precooled on ice. 17. 40% Glycerol stock solution. Autoclave. Dilute to 10% in distilled-water for use. 18. Phosphate-buffered saline (PBS). 19. Glass beads, 425–600-µm diameter, acid-washed. 20. Lysis buffer: 4% (w/v) sodium dodecyl sulfate (SDS), 20% (v/v) glycerol, 0.005% (w/v) bromophenol blue, 0.1 M Tris-HCl, pH 6.8, 280 mM β-mercaptoethanol. Dissolve 2 g SDS, 10 g glycerol, and 0.0025 g bromophenol blue in 25 mL 0.5 M Tris-HCl, pH 6.8. Adjust the volume to 50 mL. Just before use, add 20 µL β-mercaptoethanol to 1 mL of lysis buffer. 21. 14% SDS-polyacrylamide gel electrophoresis (PAGE) gel and apparatus; Coo- massie blue staining solution. 3. Method 3.1. Transformation of Yeast Strains Yeast strains can be transformed by electroporation (7) or LiAC (8). Elec- troporation is preferable because it gives higher transformation effi ciencies. 3.1.1. Transformation of Yeast by LiAC Method 1. Inoculate a colony of the yeast strain of choice (e.g., SU51) into 5 mL selective SD broth (SD broth supplemented with glucose, leucine, and histidine for SU51), and grow with agitation overnight at 30°C. 2. Inoculate 50 mL YEPD broth with approx 50 µL (see Note 4) of the overnight culture and grow with agitation overnight at 30°C. 3. Harvest the yeast cells when the culture reaches an optical density 660 nm (OD 660 ) of 1.0–2.0. Centrifuge the culture for 5 min at 4000g. 4. Pour off the growth medium, and discard. Resuspend the cell pellet in 25 mL sterile H 2 O and centrifuge as above. 5. Pour off the distilled water and discard. Resuspend the cells in 1.0 mL 100 mM LiAc and transfer the suspension to a 1.5 mL sterile microtube. 6. Centrifuge the suspension at 14,000g (top speed in a microcentrifuge) for 15 s, then remove the LiAC with a micropipet. 7. Resuspend the cell pellet in 100 mM LiAC to a fi nal volume of 300 µL (approx 2 × 10 9 cells/mL). 8. Pipet 50 µL samples of the cells into labeled microtubes. Centrifuge at 14,000g for 15 s and remove the LiAC. 9. Add 240 µL PEG solution to each cell pellet. Resuspend carefully, but thoroughly (see Note 5). 10. Add 36 µL 1.0 M LiAC to each suspension and mix thoroughly. 11. Add 25 µL boiled ssDNA (2.0 mg/mL) to each suspension and mix thoroughly. 362 van der Vaart 12. Add 50 µL sterile H 2 O containing plasmid DNA (0.1–10 µg) and mix thoroughly. 13. Incubate the microtubes for 30 min at 30°C. 14. Heat-shock the yeast cells by incubating in a water bath at 42°C for 20–25 min (see Note 6). 15. Centrifuge the microtubes at 6000–8000g for 15 s and discard the supernatant. 16. Pipet 250 µL sterile H 2 O into each microtube and resuspend the pellet gently. 17. Plate the cell suspension on the appropriate SD or YEPD agar plates (for the SU51 strain transformed with a plasmid containing the LEU2 marker gene, SD plates, supplemented with GLU and histidine, are used). 18. Incubate the agar plates for 2–4 d at 30°C to recover transformants. 3.1.2. Transformation of Yeast by Electroporation For the generation of yeast cells with a high transformation effi ciency, it is essential to use precooled buffers, and to keep the yeast cells on ice throughout this protocol (except where stated otherwise). 1. Grow the yeast strain as described in Subheading 3.1.1., except inoculate 150 mL YEPD broth. 2. Harvest the culture when it reaches an OD 660 of 1.0–2.0 by centrifugation at 4000g for 5 min. 3. Discard the growth medium and resuspend the cell pellet in 2.0 mL HEPES/ dithiothreitol buffer and transfer the suspension to two sterile microtubes. 4. Incubate both microtubes in a water bath at 30°C for 10 min (see Note 7). 5. Centrifuge the suspensions at 14,000g for 15 s and remove the supernatant. 6. Resuspend each cell pellet in 1 mL ice-cold distilled water and incubate on ice for 2–3 min. 7. Centrifuge as in step 5 and remove the supernatant. 8. Repeat steps 6 and 7. 9. Resuspend each cell pellet in 1 mL ice-cold 10% glycerol and incubate on ice for 2–3 min. 10. Centrifuge as in step 5 and remove the supernatant. 11. Repeat steps 9 and 10. 12. Resuspend the cells in ice cold 10% glycerol, according to the following equation: Final volume (mL) = OD 660 of the initial culture/1.46 The fi nal volume should be approx 1–1.5 mL/microtube. 13. Keep the cells on ice for at least 1 h before electroporation. 14. Mix 50 µL aliquots of the competent cells with the appropriate amount of DNA (3–5 µg of an 8 kb plasmid) (see Note 8). 15. Transfer the mixture to a precooled electrocuvet and electroporate at 800 Ω, 25 µF and at the following voltages: 0.9, 1.0, 1.1, and 1.2 kV. Time constants should be 10–16 ms (see Note 9). Expression of V HH Ab Fragments 363 16. Quickly add 0.8 mL prewarmed YEPD broth (30°C) to the electroporated cells and incubate at 30°C for 1 h without shaking. 17. Plate the cells on plates and incubate as described in Subheading 3.1.1. (see Note 10). 3.2. Growth and Induction of Yeast Transformants 1. Pick several yeast transformants, and strike them out on selective SD plates. 2. Pick a single colony and grow overnight with agitation in 3 mL selective SD broth at 30°C. 3. Inoculate a 1Ϻ100 dilution of the culture in 10 mL YEPD induction medium (see Note 11), and grow overnight at 30°C (see Note 12). The remaining culture is used to prepare glycerol stocks: mix 0.8 mL culture with 0.8 mL 40% sterile glycerol, and store at –80°C (see Note 13). 4. Transfer 1 mL culture to a microtube and centrifuge at 14,000g for 15 s. 5. Place the supernatant in fresh microtube (i.e., medium fraction). 6. Resuspend the cell pellet in 1 mL PBS and centrifuge at 14,000g for 15 s. 7. Discard the supernatant and resuspend the cell pellet in 0.5 mL PBS. 8. Add 1 g glassbeads to the cell suspension. 9. Lyse the yeast cells by vortexing 4× for 30 s. Keep the cell suspension on ice between vortexing. 10. Add 0.5 mL PBS and transfer the supernatant fraction to a fresh microtube. 11. Centrifuge the suspension for 15 min at 14,000g at 4°C. 12. Transfer the supernatant (i.e., soluble cell fraction) into a fresh microtube. 13. Resuspend the pellet in 500 µL PBS (i.e., insoluble cell fraction). 14. Add 15 µL lysis buffer to 15 µL of each fraction (steps 5, 12, and 13) and boil for 5 min. 15. Electrophorese the samples on a 14% SDS-PAGE gel to separate the proteins. Visualize the protein bands by Coomassie blue staining or by Western blot analysis with appropriate Abs. 16. The fractions can then be analyzed by enzyme-linked immunosorbant assay or other appropriate assay to analyze the Ab fragment specifi city and/or functionality. 17. Samples can be stored at –20°C. Further purifi cation of Ab fragments can be performed by ion exchange chromatography or by Protein A purifi cation (the latter is only applicable if the Ab fragment binds to Protein A). 4. Notes 1. Auxotrophic or dominant markers can be used as selection markers on plasmids. Auxotrophic markers complement a defi ciency (e.g., LEU2, complementing leucine defi ciency, or HIS4, complementing histidine defi ciency). Dominant markers introduce resistance against a harmful compound (e.g., resistance against geneticin or chloramphenicol). To establish secretion of an Ab fragment expressed in yeast, a signal sequence needs to be included at the N-terminus of 364 van der Vaart the Ab fragment coding sequence. For secretion of llama V HH , we use either the signal sequence of the SUC2 gene (encoded in plasmid pUR4548) or the signal sequence of the mating factor α gene. 2. Plasmid pUR4548 can also be used for the expression of scFvs, but the yield is much lower than that of V HH (heavy chain only). 3. It is not necessary or desirable to boil the carrier DNA every time. After boiling, it is best to keep a small aliquot in a freezer box and boil again only after 3–4 freeze-thaws. 4. Yeast strains differ in growth rate. This rate determines the dilution by which yeast strains are inoculated in YEPD before transformation. On average, yeast strains used in laboratories will need between 15 and 20 h after inoculation to reach this OD. 5. The resuspension of yeast cells in PEG should be performed gently, but thor- oughly. Cells that are not dissolved properly are not shielded from the detrimental effects of the high concentration of LiAC. 6. The optimum time for heat shock may vary for different yeast strains and may need to be tested to obtain a high effi ciency. 7. During this step the yeast cells are producing CO 2 . Make sure that the lids of the microtubes do not snap open because of build-up of pressure during the incubation by securing the lids, e.g., with a weight. Release the pressure before the centrifugation step, by opening the microtubes. 8. The amount of DNA that is added to the yeast cells is dependent on the size of the plasmid that has to be transformed. For smaller plasmids, less DNA will be necessary for an effi cient transformation. 9. Depending on the yeast strain used, the optimal voltage for the most effi cient transformation effi ciency has to be determined. For subsequent transformations, only the most optimal voltage is used (for SU51, this is 1.0 kV). 10. When generation of the transformants is on minimal medium plates, a wash in 1 M sorbitol is recommended (centrifuge the electroporated cells at 14,000g for 15 s, remove the supernatant, add 1 mL 1 M sorbitol, then resuspend the cells carefully). If washing is omitted, a slightly more intensive background of nontransformed cells is visible. 11. The type of induction medium that is used for production of an Ab fragment in yeast is dependent on the promoter that is placed in front of the Ab fragment gene. If a constitutive promoter is used (e.g., PGK), no additives are needed for promoter activation (just a carbon source for yeast growth is suffi cient). In case of an inducible promoter, a compound must be added to the growth medium for promoter activation. In case of GAL7, 2.5% w/v galactose is added (the addition of glucose will repress this promoter). 12. Optionally, the cultures can be grown in induction medium for an additional 24 h. Depending on the yeast strain and the expression system used, this can result in higher protein yields (for SU51 transformed with pUR4548, 24 h induction is suffi cient). Expression of V HH Ab Fragments 365 13. Transformants can be recovered from glycerol stocks by striking them out on selective SD plates, and growning as described in Subheading 3.2. The stability of the yeast transformants is not decreased during storage. References 1. Horwitz, A. H., Chang, C. P., Better, M., Hellstrom, K. E., and Robinson, R. R. (1988) Secretion of functional antibody and Fab fragment from yeast-cells. Proc. Natl Acad. Sci. USA 85, 8678–8682. 2. Shusta, E. V., Raines, R. T., Pluckthun, A., and Wittrup, K. D. (1998) Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments. Nature Biotechnol. 16, 773–777. 3. Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G., Hamers, C., Songa, E. B., Bendahman, N., and Hamers, R. (1993) Naturally-occurring antibodies devoid of light-chains. Nature 363, 446–448. 4. Frenken, L. G. J., van der Linden, R. H. J., Hermans, P. W. J. J., Bos, J. W., Ruuls, R. C., de Geus, B., and Verrips, C. T. (2000) Isolation of antigen specifi c llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae. J. Biotech. 78, 11–21. 5. Romanos, M. A., Scorer, C. A., and Clare, J. J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423–488. 6. Van der Vaart, J. M., de Biesebeke, R, Chapman, J. W., Toschka, H. Y., Klis, F. M., and Verrips, C. T. (1997) Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous proteins. Appl. Env. Microb. 63, 615–620. 7. Weaver, J. C., Harrison, G. I., Bliss, J. G., Mourant, J. R., and Powell, K. T. (1988) Electroporation: high-frequency of occurrence of a transient high-permeability state in erythrocytes and intact yeast. FEBS Lett. 229, 30–34. 8. Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) Transformation of intact yeast-cells treated with alkali cations. J. Bacteriol. 153, 163–168. 366 van der Vaart 367 From: Methods in Molecular Biology, vol. 178: Antibody Phage Display: Methods and Protocols Edited by: P. M. O’Brien and R. Aitken © Humana Press Inc., Totowa, NJ 33 Intrabodies Targeting scFv Expression to Eukaryotic Intracellular Compartments Pascale A. Cohen 1. Introduction Molecular techniques for inhibiting the expression of specifi c genes represent a highly refined approach to the analysis and manipulation of microbial and cellular pathways. The specifi c and high affi nity binding properties of antibodies (Abs), combined with their ability to be stably expressed in precise intracellular locations inside mammalian cells, have provided a powerful new family of molecules for gene therapy. These intracellular Abs are called “intrabodies.” A key factor contributing to the success of this approach has been the use of single-chain Abs (scFvs) in which the heavy- and light-chain variable domains (V H and V L , respectively) are synthesized as a single polypeptide, and are separated by a fl exible linker peptide, generally (GGGGS) 3 . The result is a small molecule of approx 28 kDa. Examples of Fab intrabodies have also been reported, but only where an internal ribosomal entry site has been used to allow stoichiometric amounts of heavy- and light-chain fragments to be expressed simultaneously (1,2). Intrabodies can be directed to cellular compartments such as the cytoplasm, endoplasmic reticulum (ER), nucleus, or mitochondria, by modifi cation with N-terminal or C-terminal extensions that encode classic intracellular-traffi ck- ing signals. Once the targeted compartment is reached, intrabodies can modulate cellular physiology and metabolism by a wide variety of mechanisms. They may block or stabilize macromolecular interactions, such as protein– protein or protein–DNA interactions; they may modulate enzyme function by Intrabodies 367 occluding an active site, sequestering substrate, or fi xing the enzyme in an active or inactive conformation; they may divert proteins from their usual cellular compartment, for example, by sequestering transcription factors in the cytosol or by retention in the ER of proteins that are destined for the cell surface or secretion pathways (hormones, cytokines, or surface molecules). Intracellular Ab effi cacy has been demonstrated in studies on human immunodefi ciency virus 1 (HIV-1) infection (3,4), and on oncogene or tumor suppressor protein functions (5–7), showing their potential value in gene therapy. Intrabodies intended for localization in the ER are generally fi tted with a leader peptide and the ER retention signal, KDEL, at their carboxy-termini. This peptide sequence corresponds to the carboxy-terminus of the BiP protein (8). A KDEL-tagged scFv intrabody has been used to downregulate the α-subunit of the receptor for human interleukin (IL2) and to immunomodulate interleukin receptor-dependent tumor cell growth (9). Moreover, ER-targeted scFv intrabodies have been shown to decrease markedly the cell surface expression of human and rhesus CCR5-dependent HIV-1 and simian immuno- defi ciency virus envelope glycoprotein, preventing CCR5-dependent HIV-1 infection (4). Although scFvs are small enough to pass through nuclear pores, the addition of a nuclear localization signal (NLS) increases their transport effi ciency. The most common NLS used for nuclear targeting of intrabodies is PPKKKRKV from the large T antigen of SV40. As an example, a PPKKKRKV-scFv has been designed to modulate HIV-1 Tat-mediated LTR transactivation (10). Directing scFv to mitochondria has also been described (11), and can be achieved by ligation of the N-terminal presequence of subunit VIII of human cytochrome c oxidase (COX8.21) in frame with the scFv. Intrabody expression in the cytosol has generally been accomplished by simple removal of the immunoglobulin (Ig) leader sequences. However, folding and stability problems often occur, resulting in low expression levels, limited half-life, and formation of insoluble aggregates (12). This is probably caused by the reducing environment of the cell cytoplasm, which hinders the formation of the intrachain disulfi de bond of the V H and V L domains, important for the stability of the folded protein. Because many residues in the frameworks contribute to the folding stability of Ab domains (13), different scFvs will have different overall stability. Therefore, those scFvs that are intrinsically more stable will tolerate the loss of the intrachain disulfi de bonds and remain folded, but others will not. Different studies (5,10) have pointed out that fusing a κ-chain constant domain (C κ ) at the carboxy-terminus of the scFv cassette (scFv–C κ ) may increase the stability of scFvs expressed in the cytosol possibly by a dimerization event. Recently, different groups have applied methods of 368 Cohen evolutionary engineering to the generation of functional intrabodies. One group (13) has used random mutagenesis and screening; others (14) have engineered stabilizing mutations predicted from a consensus sequence analysis or used a two-hybrid in vivo system to select functional intracellular Abs (15). Unfortunately, the general application of these methods does not appear to be straightforward. Targeting Ig expression to eukaryotic intracellular compartments comprises the following steps: 1. Cloning the V H and V L domains of interest and engineering the corresponding scFv. Insertion into a prokaryotic vector and expression in Escherichia coli to investigate the scFv’s functionality, in terms of folding and binding to the antigen. Most of the described scFvs arise from well-characterized murine hybridomas. 2. Addition of N- or C-terminal extensions that encode classical intracellular- traffi cking signals to target the recombinant Ab to the intended cellular compartment. 3. Insertion of the modifi ed scFv into a eukaryotic expression vector, then transfec- tion of mammalian cells. Investigation by in vitro studies of stability, localization, and binding of the expressed scFv to the antigen of interest. In this chapter, we focus on the construction of both nuclear- and cytosol- targeted scFvs, on the assumption that construction and prokaryotic expression have already been achieved. We also describe transfection of eukaryotic cells with these constructs and immunofl uorescence detection in the intracellular environment. 2. Materials 1. An scFv cloned into an appropriate prokaryotic expression vector. This protocol describes the anti-p53 scFv DO-1 cloned into the pCANTAB5E phagemid (Pharmacia Biotech, Uppsala, Sweden). 2. Ultrapure-grade H 2 O (MilliQ or equivalent). Autoclave. 3. Stock solution of 100 mM deoxyribonucleoside triphosphates (dNTPs). Keep at –20°C. Make a 2 mM solution by dilution with sterile H 2 O. 4. AmpliTaq DNA Polymerase 5 U/µL (Perkin-Elmer, Gaithersburg, MD). Store at –20°C in a constant-temperature freezer. The enzyme is provided with a 25 mM MgCl 2 solution and a 10X PCR buffer II (100 mM Tris-HCl, pH 8.3, 500 mM KCl). 5. Oligonucleotide primers: these can be ordered from any oligonucleotide synthesis company. Store at –20°C. 6. Thermocycler (Perkin-Elmer, Gene Amp PCR system). 7. Kit for the isolation of polymerase chain reaction (PCR) products (e.g., Wizard PCR preps system, Promega, Madison, WI). Keep at room temperature. Intrabodies 369 [...]... with 5% FCS and antibiotics 16 DMEM containing 5% FCS and 800 µg/mL zeocin (Invitrogen) 17 6-, 2 4-, and 96-Well sterile tissue culture plates; fluorescence-activated cellsorting (FACS) tubes (Greiner) scFvs in Eukaryotic Cells 383 3 Methods 3.1 Subcloning of scFv into pLead-mycHis 1 Isolate the scFv DNA from the phage- display expression vector using SfiI and NotI, and subclone into the pLead-mycHis vector... Hooijberg, E., Brakenhoff, R H., Meulen-Muileman, I H., Pinedo, H M., and Boven, E (1998) Construction and characterization of a fusion protein of single-chain anti-CD20 antibody and human beta-glucuronidase for antibody- directed enzyme prodrug therapy Blood 92, 184–190 2 Yokota, R., Milenic, D E., Whitlow, M., and Schlom, J (1992) Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin... Leek, The Netherlands) This plasmid allows eukaryotic expression under the control of a cytomegalovirus promoter 9 EcoRI restriction enzyme (20 U/µL) and XbaI restriction enzyme (20 U/µL) with bovine serum albumin (BSA) 100 X and NEBuffer 2 10X (500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, 10 mM dithiothreitol, pH 7.9) (New England Biolabs, Beverly, MA) Keep at –20°C 10 DNA Mass Ladder (Gibco-BRL, Gaithersburg,... A., and Marasco, W A (1995) Phenotypic knock-out of the high-affinity human interleukin 2 receptor by intracellular single-chain antibodies against the alpha subunit of the receptor Proc Natl Acad Sci USA 92, 3137–3141 10 Mhashilkar, M., Bagley, J., Chen, S Y., Szilvay, A M., Helland, D G., and Marasco, W A (1995) Inhibition of HIV-1 tat-mediated LTR transactivation and HIV-1 infection by anti-tat... Molecular Biology, vol 178: Antibody Phage Display: Methods and Protocols Edited by: P M O’Brien and R Aitken © Humana Press Inc., Totowa, NJ 389 390 Sanna Fig 1 Map of the expression vector pFab–CMV showing functional regions and unique restriction sites for cloning of HC and LC and excision of Ig γ1 constant-region sequences these HC constant regions by digestion with NheI and SpeI, brings a second... Molecular Biology, vol 178: Antibody Phage Display: Methods and Protocols Edited by: P M O’Brien and R Aitken © Humana Press Inc., Totowa, NJ 379 380 de Graaf et al Fig 1 Schematic representation of the expression cassette of the eukaryotic vector pLead-mycHis The structural elements include the CMV promoter, immunoglobulin κ leader (secretion) sequence, C-terminal myc and 6His tags, and a MCS The MCS contains... transduced with an AAV vector expressing a human anti-gp120 antibody Hum Gene Ther 7, 1515–1525 3 Marasco, W A., LaVecchio, J., and Winkler, A (1999) Human anti-HIV-1 tat scFv intrabodies for gene therapy of advanced HIV-1-infections and AIDS J Immunol Methods 231, 223–238 4 Steinberger, P., Andris-Widhopf, J., Buhler, B., Torbett, B E., and Barbas, C F., 3rd (2000) Functional deletion of the CCR5... Fragments and Whole Ig 391 3 Restriction and modifying enzymes and buffers: XhoI, SacI, SpeI, XbaI, NheI; T4 DNA ligase 4 Competent Escherichia coli cells 5 Bacterial media and supplements for selection: Luria-Bertani (LB) liquid and solid media; sterile ampicillin stock solution (25 mg/mL in H2O); LB-agar plates containing 100 µg/mL ampicillin (LBA) 6 Purification kits for small- and large-scale extraction... J., and Marasco, W A (1994) Combined intra- and extracellular immunization against human immunodeficiency virus type 1 infection with a human anti-gp120 antibody Proc Natl Acad Sci USA 91, 5932–5936 2 Chen, J D., Yang, Q., Yang, A G., Marasco, W A., and Chen, S Y (1996) Intra- and extracellular immunization against HIV-1 infection with lymphocytes transduced with an AAV vector expressing a human anti-gp120... Abs: anti-myc 9E10 (10) (supernatant from the hybridoma cell line [American Type Culture Collection]), horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin G (Dako); fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin G (Dako) 9 AEC horseradish peroxidase substrate (Dako) 10 Buffers, apparatus, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . Dissolve 15 g Bacto-agar in 790 mL distilled water and autoclave. Cool to 60°C and add 100 mL 10X stock of yeast nitrogen base without amino acids, 100 mL 10X glucose stock, and 10 mL 100 X stock(s). (BSA) 100 X and NEBuffer 2 10X (500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl 2 , 10 mM dithiothreitol, pH 7.9) (New England Biolabs, Beverly, MA). Keep at –20°C. 10. DNA Mass Ladder (Gibco-BRL,. LaVecchio, J., and Winkler, A. (1999) Human anti-HIV-1 tat scFv intrabodies for gene therapy of advanced HIV-1-infections and AIDS. J. Immunol. Methods 231, 223–238. 4. Steinberger, P., Andris-Widhopf,