Signal Sequence Trap

Expression Cloning Method for Secreted Proteins and Type 1 Membrane Proteins
  • Kei Tashiro
  • Toru Nakano
  • Tasuku Honjo
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 69)

Abstract

Intercellular signaling and cell adhesion are among the most critical mechanisms for development and maintenance of multicellular organisms. Although a large number of molecules involved in signaling or adhesion have been cloned, there still remain many unknown molecules that are important in these functions. Most of the molecules involved in signaling or adhesion are secreted or membrane-anchored proteins. Many of these proteins contain a signal sequence or leader peptide in the N-terminal of their premature form (1), The traditional strategy for cloning these genes requires a functional assay to detect the specific function of each molecule. To establish a general cDNA cloning method for growth factors, hormones, neuropeptides, their receptors, and adhesion molecules, we have developed a new cloning strategy for selecting cDNA fragments encoding N-terminal signal sequences (2). This method, called Signal Sequence Trap, turns out to be an efficient method to isolate 5′-cDNA fragments from secreted or transmembrane proteins. We have already obtained a number of cDNA clones of putative growth factors, receptors, or adhesion molecules (2, 3, 4, 5). Signal Sequence Trap can clone not only secreted proteins, GPI-anchored proteins, and plasma membrane proteins, but also proteins located in endoplasmic reticulum (ER), Golgi apparatus (GA), and lysosome.

1 Introduction

Intercellular signaling and cell adhesion are among the most critical mechanisms for development and maintenance of multicellular organisms. Although a large number of molecules involved in signaling or adhesion have been cloned, there still remain many unknown molecules that are important in these functions. Most of the molecules involved in signaling or adhesion are secreted or membrane-anchored proteins. Many of these proteins contain a signal sequence or leader peptide in the N-terminal of their premature form (1), The traditional strategy for cloning these genes requires a functional assay to detect the specific function of each molecule. To establish a general cDNA cloning method for growth factors, hormones, neuropeptides, their receptors, and adhesion molecules, we have developed a new cloning strategy for selecting cDNA fragments encoding N-terminal signal sequences (2). This method, called Signal Sequence Trap, turns out to be an efficient method to isolate 5′-cDNA fragments from secreted or transmembrane proteins. We have already obtained a number of cDNA clones of putative growth factors, receptors, or adhesion molecules (2, 3, 4, 5). Signal Sequence Trap can clone not only secreted proteins, GPI-anchored proteins, and plasma membrane proteins, but also proteins located in endoplasmic reticulum (ER), Golgi apparatus (GA), and lysosome.

The epitope-tagging expression plasmid vector used in Signal Sequence. Trap is the pcDLSRα-Tac(3′) vector (2,6), which directs the cell-surface expression of Tac (human CD25, a chain of the human interleukin-2 receptor) fusion proteins when inserts with N-terminal signal sequences are cloned in-frame, in the correct orientation (7) (seeFig. 1). The Tac epitope-tagged fusion protein expressed on plasma membranes is easily detected with antihuman CD25 antibodies (8). The first strand of cDNA is synthesized with random hexamers, deoxycytosine (dC) tails are added at the 3′-end of the first-strand cDNA, and second-strand synthesis is carried out with an EcoRI-linker primer that contains polydeoxyguanosine (dG). The product is ligated with a SacI adapter, size-fractionated to 300–500 bp by agarose gel electrophoresis (seeNote 1), and amplified by polymerase chain reaction (PCR) (9). The amplified fragments are digested with EcoRI and SacI, size-fractionated by agarose gel electrophoresis again, and inserted into the pcDL-SRa-Tac(3′) vector in the same orientation with the Tac cDNA. Using the resulting expression library, sib-screening should be done (see ref. 10 and Note 2) (seeFig. 2). After Escherichia coli transformation, 49 individual colonies are plated on a 9-cm agar plate in a matrix format (seven rows by seven lines) and assigned to one pool. COS-7 cells are transfected with plasmid DNAs of each pool (11), and fusion proteins expressed on the cell surface are microscopically detected by immunostaining with antihuman CD25 antibodies. If a pool contains a positive clone plasmid, 14 smaller pools consisting of seven individual clones in each row or line of the matrix are tested until a single positive clone can be determined. The nucleotide sequence information of the positive clones is used to identify hydrophobic amino acid residues, which are the core of N-terminal signal sequence. Also, a homology search in the database is done to compare the sequence with known proteins. With stringent hybridization condition, the full-length cDNA clones can be obtained by using trapped cDNA fragment encoding the N-terminal signal sequence to probe an oligo(dT)-primed cDNA library.
Fig. 1.

Schematic diagram of signal sequence trap.

Fig. 2.

Schematic view of sib-screening.

2 Materials

2.1 First-Strand Synthesis

  1. 1.

    PolyA+ RNA extracted from the cells or tissues of interest.

     
  2. 2.

    Random hexamer DNA (Takara, Kyoto, Japan).

     
  3. 3.

    DEPC-treated H2O.

     
  4. 4.

    5X Reverse transcription buffer 0.25M Tris-HCl, pH 8.3, 0.375M KCl, 15 mM Mgcl2.

     
  5. 5.

    0.1M Dithiothreitol(DDT).

     
  6. 6.

    5 mM dNTP: 5 mM dATP, 5 mM dTTP, 5 mM dCTP, 5 mM dGTP.

     
  7. 7.

    Super Script II, (Gibco BRL, Gaithersburg, MD), 200 U/µL.

     
  8. 8.

    0.5M EDTA.

     
  9. 9.

    1N NaOH.

     
  10. 10.

    2M Tris-HCl, pH 7.4.

     
  11. 11.

    1N HCl.

     
  12. 12.

    TE: 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA.

     
  13. 13.

    10M Ammonium acetate.

     
  14. 14.

    10 µg/µL Glycogen.

     
  15. 15.

    Phenol/CIAA: 25∶24∶1 phenol equilibrated with 0.1M Tris-HCI, pH. 8.0/chloroform:isoamyl alcohol.

     
  16. 16.

    Ethanol.

     

2.2 dC-Tailing and Second-Strand Synthesis

  1. 1.

    1 mMdCTP.

     
  2. 2.

    5X Reverse transcription buffer: 0.25M Tris-HCl, pH 8.3, 0.375M KCl, 15 mM MgCl2 (for dC-Tailing, final concentration is 1/2X, not 1X).

     
  3. 3.

    Terminal deoxynucleotidyl transferase (Stratagene, La Jolla, CA), 20 U/mL

     
  4. 4.

    TE: 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA.

     
  5. 5.

    3M Sodium acetate.

     
  6. 6.

    Phenol/CIAA: 25∶24∶1 phenol equilibrated with 0.1M Tris-HCl, pH 8.0/chloroform:isoamyl alcohol.

     
  7. 7.

    10 µg/µL Glycogen.

     
  8. 8.

    Ethanol.

     
  9. 9.

    25 µM ESLG primer: 5′-GCGGCCGCGAATTCTGACTAACTGAC(G)17.

     
  10. 10.

    5X Reverse transcription buffer: 0.25M Tris-HCl, pH 8.3, 0.375M KCl, 15 mM MgCl2.

     
  11. 11.

    5 mM dNTP: 5 mM dATP, 5 mM dTTP, 5 mM dCTP. 5 mM dGTP.

     
  12. 12.

    0.1M DTT.

     
  13. 13.

    200 U/µL Super script II (Gibco BRL, Gaithersburg, MD).

     

2.3 SacI Adapter Ligation and Size Selection

  1. 1.

    25 µM SSEH oligomer DNA: S′-CCGCGAGCTCGATATCAAGCTTGTAC.

     
  2. 2.

    1OX Kination buffer: 700 mM Tris-HCl, pH 7.6, 100 mM MgCl2, 50 mM DTT.

     
  3. 3.

    5 mM dATP.

     
  4. 4.

    10 U/µL T4 Polynucleotide kinase. (New England Biolabs).

     
  5. 5.

    Phenol/CIAA: 25∶24∶1 phenol equilibrated with 0.1M Tris-HCl, pH 8.0/chloroform/isoamyl alcohol.

     
  6. 6.

    3M Sodium acetate.

     
  7. 7.

    Ethanol.

     
  8. 8.

    25 µM LLHES oligomer DNA: 5′-GAGGTACAAGCTTGATATCGAGCTCGCGG.

     
  9. 9.

    10X Ligation buffer: 500 mM Tris-HCl, pH 7.8, 100 mM MgCl2, 100 mM DTT, 10 mM adenosine triphosphate (ATP), 250 µg/mL BSA.

     
  10. 10.

    400 U/µmL T4 DNA ligase (New England Biolabs).

     
  11. 11.

    Agarose.

     
  12. 12.

    1X TAE: 0.04M Tris-acetate, 1 mM EDTA.

     
  13. 13.

    10 mg/mL Ethidium bromide.

     
  14. 14.

    6X Loading buffer 0.25% bromophenol blue, 30% glycerol in H2O.

     
  15. 15.

    DNA size markers.

     
  16. 16.

    The GeneClean II Kit (BIO 101 Inc., La Jolla, CA), containing 6M sodium iodite, Glassmilk and New Wash.

     
  17. 17.

    TE: 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA.

     

2.4 PCR Amplification

  1. 1.

    Thermal cycler.

     
  2. 2.

    10X PCR buffer: 100 mM Tris-HCl, pH 8.3, 500 mM KC1, 15 mM MgCl2 0.01% (w/v) gelatin.

     
  3. 3.

    5 mM dNTP: 5 mM dATP, 5 mM dTTP, 5 mM dCTP, 5 mM dGTP.

     
  4. 4.

    25 µM ESL oligomer DNA: 5′-GCCGCGAATTCTGACTAACTGAC.

     
  5. 5.

    25 µM LLHES oligomer DNA:5′-GAGGTACAAGCTTGATATCGAGCTCGCGG.

     
  6. 6.

    5 U/µL Taq DNA polymerase (Takara).

     
  7. 7.

    Mineral oil.

     
  8. 8.

    Agarose.

     
  9. 9.

    1X TAE: 0.04M Tris-acetate, 1 mM EDTA.

     
  10. 10.

    10 ng/mL Ethidium bromide.

     
  11. 11.

    6X Loading buffer: 0.25% bromophenol blue, 30% glycerol in H2O.

     
  12. 12.

    DNA size markers.

     

2.5 Insert CDNA Digestion with SacI and EcoRI

  1. 1.

    10X Low-salt restriction enzyme buffer: 100 mM Tris-HCl, pH 7.5, 100 mM MgCl2, 10 mM DTT.

     
  2. 2.

    10 U/µL SacI.

     
  3. 3.

    1OX Medium-salt restriction enzyme buffer. 100 mM Tris-HCl, pH 7.5, 100 mM MgCl2, 10 mM DTT, 500 mM NaCl.

     
  4. 4.

    10 U/µL EcoRI.

     
  5. 5.

    Agarose.

     
  6. 6.

    1X TAE: 0.04M Tris-acetate, 1 mM EDTA.

     
  7. 7.

    10 ng/mL Ethidium bromide.

     
  8. 8.

    6X Loading buffer: 0.25% bromophenol blue, 30% glycerol in H2O.

     
  9. 9.

    DNA size markers.

     
  10. 10.

    TE: 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA.

     

2.6 Vector Preparation

  1. 1.

    10X Low-salt restriction enzyme buffer: 100 mM Tris-HCl, pH 7.5, 100 mM MgCl2, 10 mM DTT.

     
  2. 2.

    pcDL-SRα-hRAR(S′)-Tac(3′): a negative control plasmid; it has 5′-portion of human retinoic acid receptor, which does not bear N-terminal signal sequence (2). It can be obtained from Tasuku Honjo (Department of Medical Chemistry, Kyoto University, Kyoto, Japan).

     
  3. 3.

    10 U/µL SacI.

     
  4. 4.

    1OX Medium-salt restriction enzyme buffer: 100 mM Tris-HCl, pH 7.5, 100 mM MgCl2, 10 mM DTT, 500 mM NaCl.

     
  5. 5.

    10 U/µL EcoRI.

     
  6. 6.

    Agarose.

     
  7. 7.

    1X TAE. 0.04M Tris-acetate, 1 mM EDTA.

     
  8. 8.

    10 ng/mL Ethidium bromide.

     
  9. 9.

    6X Loading buffer: 0.25% bromophenol blue, 30% glycerol in H2O.

     
  10. 10.

    DNA size markers.

     
  11. 11.

    TE: 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA.

     

2.7 Vector-Insert Ligation

  1. 1.

    1OX Ligation buffer: 500 mM Tris-HCl, pH 7.8, 100 mM MgCl2, 100 mM DTT, 10 mM ATP, 250 µg/mL BSA.

     
  2. 2.

    400 U/µL T4 DNA ligase (New England Biolabs).

     

2.8 Transformation

  1. 1.

    XL-1 Blue competent cells (Stratagene).

     
  2. 2.

    SOC medium.

     
  3. 3.

    LB agar plates containing 100 µ/mL ampicillin.

     

2.9 Making Pool Plate and DNA Preparation

  1. 1.

    LB agar plates containing 100 µg/mL ampicillin.

     
  2. 2.

    Terrific broth containing 100 µg/mL ampicillin (10).

     
  3. 3.

    Autoclaved toothpicks.

     
  4. 4.

    GTE buffer: 50 mM glucose, 25 mM Tris-HCl, pH 8.0, 10 mM EDTA (pH 8.0).

     
  5. 5.

    0.2N NaOH, 1% sodium dodecyl sulfate (SDS).

     
  6. 6.

    5M Potassium acetate.

     
  7. 7.

    5M Lithium chloride.

     
  8. 8.

    Isopropanol.

     
  9. 9.

    Ethanol.

     
  10. 10.

    100 µg/µL RNase A.

     
  11. 11.

    Phenol/CIAA: 25∶24∶1 phenol equilibrated with 0.1M Tris-HCl, pH 8.0/chloroform/isoamyl alcohol.

     
  12. 12.

    10 µg/µL Glycogen.

     
  13. 13.

    3M Sodium acetate.

     
  14. 14.

    pcDL-SRα-hRAR(5′)-Tac(3′).

     
  15. 15.

    pcDL-SRα-hG-CSF(5′)-Tac(3′): A positive control plasmid; it has a 5′-portion of human G-CSF, which has an N-terminal signal sequence (1). It can be obtained from Tasuku Honjo.

     

2.10 Transfection into COS-7 Cells

  1. 1.

    COS-7 cells (can be obtained from American Type Culture Collection [ATCC], Rockville, MD).

     
  2. 2.

    Phosphate-buffered saline (PBS) (−).

     
  3. 3.

    0.05% (w/v) Trypsin-0.02% (w/v) EDTA-PBS (−).

     
  4. 4.

    Dulbecco’s modified Eagle’s medium (DMEM) (+): supplemented with 10% (v/v) fetal calf serum.

     
  5. 5.

    DMEM (−).

     
  6. 6.

    1M Tris-HCl (pH 7.4).

     
  7. 7.

    10 mg/mL DEAE-dextran.

     
  8. 8.

    10 mM Chloroquine.

     
  9. 9.

    10% (v/v) dimethyl sulfoxide (DMSO).

     

2.11 Cell Surface Immunostaining with Anti-Tac

  1. 1.

    Fluorescein (FITC)-conjugated antihuman CD25 (Dako, Carpinteria, CA).

     
  2. 2.

    PBS(−).

     
  3. 3.

    Fetal calf serum.

     
  4. 4.

    0.02% (w/v) EDTA in PBS (−).

     

2.12 Making Smaller Pools and identifying Positive Clones

Materials used here are listed in Section 2.9.2.11..

2.13 Hydropathy Analysis and Database Search

  1. 1.

    SRA primer 5′-TTTACTTCTAGGCCTGACG.

     
  2. 2.

    TAC primer 5′-CCATGGCTTTGAATGTGGCG.

     
  3. 3.

    Nucleic acid and protein analysis software (Gene Works, IntelliGenetics, Mountain View, CA).

     
  4. 4.

    A personal computer accessible to the databases that have the information of Genbank, EMBL, DDBJ, and Swiss Prot.

     

3 Methods

Section 3.1., describes the 5′ terminal-enriched cDNA library construction for Signal Sequence Trap. Section 3.2. contains the protocol of sib-screening with anti-Tac Immunostaining. Section 3.3. describes types of analysis.

3.1 SST-cDNA Library Construction

3.1.1 First-Strand Synthesis and RNA Hydrolysis

  1. 1.

    Mix, with caution, 1 µg of polyA+ RNA and 10 ng of random hexamer in DEPC-treated H2O in a final volume of 11 µL (seeNotes 35).

     
  2. 2.

    Gently stir and quick spin.

     
  3. 3.

    Incubate at 70°C for 10 min, and quickly chill on ice.

     
  4. 4.

    Add 4 µL of 5X reverse transcription buffer, 2 µL of 0.1M DTT, and 2 µL, of 5 mM dNTP mix solution, and gently mix (seeNote 6).

     
  5. 5.

    Immediately add 1 µL of Super Script II (200 U/µL), gently stir, and quick spin.

     
  6. 6.

    Incubate at 49°C for 60 min.

     
  7. 7.

    Add 1 µL of 0.5M EDTA, and gently mix.

     
  8. 8.

    Add 39 µL of H2O, 15 µL of 1N NaOH, gently mix, and quick spin.

     
  9. 9.

    Incubate at 70°C for 20 min.

     
  10. 10.

    Add 4 µL of 2M Tris-HCl, pH 7.4, 14.7 µL of 1N HCl, 115 µL of TE, 140 µL of 10M ammonium acetate, 1 µL of 10 µL/µL glycogen, vortex gently, and quick spin.

     
  11. 11.

    Add 350 µL of phenol/CIAA and vortex.

     
  12. 12.

    Spin to separate the phases, and then add 2.5 vol of ethanol to the aqueous phase.

     
  13. 13.

    Put on dry ice for 20 min, and centrifuge at the full speed for 15 min.

     
  14. 14.

    Rinse the pellets with 950 µL of 75% ethanol and air-dry.

     
  15. 15.

    Dissolve the single-stranded cDNA in 20 µL of TE.

     

3.1.2 dC-Tailing and Second-Strand Synthesis

  1. 1.

    Denature cDNA at 70°C for 5 min, and chill on ice.

     
  2. 2.

    Mix 20 µL of first-strand cDNA with 6 µL of 1 mMdCTP and 3 µL of 5X reverse transcription buffer (seeNotes 7 and 8).

     
  3. 3.

    Add 1 µL of terminal deoxynucleotidyl transferase (20 U/µL), gently stir, and quick spin.

     
  4. 4.

    Incubate at 37°C for 10 min, and then at 70°C for 15 min.

     
  5. 5.

    Add 170 µL of TE, 20 µL of 3M sodium acetate, vortex vigorously for 1 min, add 200 µL of phenol/CIAA, and vortex vigorously for 1 min (seeNotes 1 and 9).

     
  6. 6.

    Spin to separate the phases, then add 1 µL of 10 µg/µL glycogen, 40 µL of 3M sodium acetate, and 2.5 vol of ethanol to the aqueous phase.

     
  7. 7.

    Set on dry ice for 20 min, and centrifuge at the full speed for 15 min.

     
  8. 8.

    Rinse the pellets with 950 µL of 75% ethanol and air-dry.

     
  9. 9.

    Dissolve in 37 µL of H2O.

     
  10. 10.

    Add 2 µL of ESLG primer (25 µM), 10 µL of 5X reverse transcription buffer, 2 µL of 5 mM dNTP, and 6 µL of DTT, gently vortex, and quick spin (seeNote 6).

     
  11. 11.

    Incubate at 70°C for 10 min, and chill on ice.

     
  12. 12.

    Add 3 µL of Super script II (200 U/µL), gently vortex, and quick spin.

     
  13. 13.

    Incubate at 42°C for 30 min.

     
  14. 14.

    Incubate at 75°C for 15 min, and chill on ice until used in Section 3.1.3. (seeNote 9).

     

3.1.3 SacI Adapter ligation and Size Selection

  1. 1.

    Incubate 84 µL of SSEH (25 µM) at 70°C for 5 min, and then chill on ice.

     
  2. 2.

    Add 10 µL of 10X Kination buffer, 5 µL of 5 mM dATP, mix, and spin.

     
  3. 3.

    Add 2 µL of T4 polynucleotide kinase (10 U/µL), mix, and quick spin.

     
  4. 4.

    Incubate at 37°C for 30 min.

     
  5. 5.

    Incubate at 70°C for 20 min.

     
  6. 6.

    Add 100 µL of phenol/CIAA, and vortex.

     
  7. 7.

    Spin to separate the phases, and then add 10 µL of 3Msodium acetate and 2.5 vol of ethanol to the aqueous phase.

     
  8. 8.

    Set on dry ice for 20 min, and centrifuge at the full speed for 15 min.

     
  9. 9.

    Rinse the pellets with 950 µL of 75% ethanol, and air-dry.

     
  10. 10.

    Dissolve the DNA in 50 µL of H2O.

     
  11. 11.

    Measure the OD260, and adjust the DNA concentration to 25 µM.

     
  12. 12.

    Mix 80 µL of 5′-phosphorylated SSEH (25 µM), and 80 µL of LLEH (25 µM).

     
  13. 13.

    Incubate at 90°C for 5 min, and slowly cool down to 22°C, 10 µL of this solution should be used in the next step. You can keep the annealed SacI adapter at −20°C. You need not repeat previous steps every time.

     
  14. 14.

    Mix 15 µL of double-stranded cDNA, 9 µL of annealed SacI adapter, and 3 µL of 10X ligation buffer, vortex, and quick spin.

     
  15. 15.

    Add 3 µL of T4 DNA ligase (400 U/µL), mix, and quick spin.

     
  16. 16.

    Incubate at 16°C for 4–16 h.

     
  17. 17.

    Incubate at 65°C for 5 min, and chill on ice.

     
  18. 18.

    Make a 1.6% agarose/TAE gel containing 30 ng/mL ethidium bromide, and load the 25 µL of ligation solution with 6 µL of 6X loading buffer. Load DNA size markers, and be sure to keep the latter at least one slot away from the cDNA.

     
  19. 19.

    Run on the gel, and cut out required fractions (e.g., 300–-450, 450–650, 650–900 bp) (seeNote 1), put each agarose block into Eppendorf tubes, and weigh them. Agarose gel blocks in one tube should be <300 µg. If a gel is >300 µg, divide into two or more tubes (seeNote 10).

     
  20. 20.

    Add 3 vol of NaI stock solution.

     
  21. 21.

    Incubate at 55°C for 5 min and vortex. You will see the agarose gel melt and become almost transparent.

     
  22. 22.

    Add 10 µL of Glassmilk, and set on ice for 5 min.

     
  23. 23.

    Wash pellet three times with New Wash.

     
  24. 24.

    Elute DNA into 20 µL of TE. Call these DNA solution “original PCR template solutions.”

     

3.1.4 PCR Amplification

  1. 1.

    Make a series of dilutions of the original PCR template solutions (e.g., 1/10, 1/100, 1/1000) to check the quality of the library.

     
  2. 2.

    Mix 1 µL of the original or a diluted PCR template solution, 2.5 µL of 10X PCR buffer, 2 µL of 5 mM dNTP, 1 µL of ESL (25 µM), 1 µL of LLHES (25 µM), 17.2 µL of H2O, and 0.3 µL of Taq DNA polymerase (5 U/µL), the total volume will be 25 µL (seeNote 11.). Vortex and quick spin.

     
  3. 3.

    Overlay a drop of mineral oil.

     
  4. 4.

    Start the thermal cycle on a program consisting of 94°C for 4 min, followed by 25 cycles of denaturation for 30 s at 94°C, annealing for 1 min at 49°C, and synthesis for 3 min at 72°C, and then followed by synthesis for 5 min at 72°C.

     
  5. 5.

    Make a 1.6% agarose/TAE gel. Load the 8 µL of each PCR reaction products mixed with 2 µL of 6X loading buffer.

     
  6. 6.

    Take a photo and make sure smears of the DNA appear not only in the reaction using the original PCR template solution, but also in the reaction using the diluted templates (seeNote 12).

     
  7. 7.

    Cut out the agarose gel containing DNA smear generated from the original PCR template solution and recover the DNA as described in Section 3.1.3.

     
  8. 18.

    Dissolve the DNA in 8 µL of H2O.

     

3.1.5 Insert cDNA Digestion with SacI and EcoRI

  1. 1.

    Mix 8 µL of recovered DNA solution, 1 µL of 10X low-salt restriction enzyme buffer, and 1 µL of SacI (10 U/µL).

     
  2. 2.

    Incubate at 37°C for 3 h.

     
  3. 3.

    Add 2 µL of 10X medium salt restriction enzyme buffer, 7 µL of H2O, and 1 µL of EcoRI.

     
  4. 4.

    Incubate at 37°C for 3 h, then incubate at 65°C for 5 min, and quickly chill on ice.

     
  5. 5.

    Run on the 1.6% agarose/TAE gel, observe the smear of DNA and recover the DNA as described in Section 3.1.3., steps 19–24.

     
  6. 6.

    Dissolve in 8 µL of TE.

     

3.1.6 Vector Preparation

  1. 1.

    Add 3 µL of 10X low-salt restriction enzyme buffer, 3 µg of negative control plasmid, pcDL-SRα-hRAR(5′)-Tac(3′) (seeNote 13), H2O to 27 µL, mix, and quick spin Add 3 µL of Sac,I mix, and quick spin.

     
  2. 2.

    Incubate at 37°C for 3 h.

     
  3. 3.

    Add 10X medium salt restriction enzyme buffer, 21 µL of H2O, and 3 µL of EcoRI, mix, and quick spin.

     
  4. 4.

    Incubate at 37°C for 3 h, then incubate at 65°C for 5 min, and quickly chill on ice.

     
  5. 5.

    Run on the 1.0% agarose/TAE gel. You will find two bands, the 291-bp band representing the retinoic acid receptor insert and the 4.5-kb digested vector band.

     
  6. 6.

    Recover the digested vector DNA as described in Section 3.1.3..

     
  7. 7.

    Dissolve in 15 µL of TE.

     
  8. 8.

    Estimate the DNA concentration using 1 µL of DNA solution to run on the 0.9% agarose/TAE gel.

     

3.1.7 Vector-Insert Ligation

  1. 1.

    Add 1 µL of insert DNA prepared in Section 3.1.5., 100 ng of digested vector prepared in Section 3.1.6., 1 µL of 10X ligation buffer, H2O to 9 µL, and 1 µL, of T4 DNA ligase (400 U/µL), mix, and quick spin. As a control without insert DNA, add 100 ng of digested vector prepared in Section 3.1.6., 1 µL of 10X ligation buffer, H2O to 9 µL, and 1 µL of T4 DNA ligase, mix, and quick spin.

     
  2. 2.

    Incubate at 16°C for 4–16 h, and then store the ligation reaction solutions at −20°C until required for transformation.

     

3.1.8 Transformation

  1. 1.

    Thaw a 100-µL aliquot of XL-1 Blue competent cell on ice, and transfer to a Falcon 2159 tube.

     
  2. 2.

    Add 1 µL of ligation solution, and set on ice for 30 min.

     
  3. 3.

    Heat-shock at 42°C for 30 s, and then immediately replace on ice.

     
  4. 4.

    Add 1.1 mL of SOC medium, and shake at 37°C for 40 min.

     
  5. 5.

    Spread 100 µL of above mixture on 12 LB agar plates containing 100 µg/mL ampicillin.

     
  6. 6.

    Transformation efficiencies are usually in the range 0.5 × 105–1 × 106 transformants/µg of DNA.

     
  7. 7.

    Check the insert size by miniprep DNA, SacI-EcoRI digestion, and run on the 1.6% agarose/TAE gel.

     

3.2 Sib-Screening with Anti-Tac Immunostaining

3.2.1 Making Pool Plates and DNA Preparation (seeNote 2)

  1. 1.

    Draw seven rows by seven lines matrix format on the bottom surface of LB agar plates containing 100 µg/mL ampicillin as shown in Fig. 2, and assign a pool plate number. It is advisable to check the system by screening a small scale, such as 1000 clones. When 1000 clones are going to be screened, prepare 20 pool plates and 20 2-mL cultures of terrific broth containing 100 µg/mL ampicillin in a Falcon 2159 tube (seeFig. 2). One person can inoculate 1000–3000 clones on LB agar plates in the 7 × 7 format in 6 h, and can prepare 20–60 pools of plasmids on the next day.

     
  2. 2.

    Forty-nine individual colonies are picked up and inoculated onto both individual squares on the pool plate and in a 2-mL terrific broth culture, and assigned to one pool as shown in Fig. 2.

     
  3. 3.

    Set the pool plates at 37°C overnight, and let a single colony grow in a square. Forty-nine individual colonies should appear on each pool plate.

     
  4. 4.

    Shake the 2-mL culture tube at 37°C overnight.

     
  5. 5.

    Transfer 1.2 mL of the full growth bacterial culture to an Eppendorf tube, and spin at the full speed for 30 s. Remaining cultures should be kept at 4°C.

     
  6. 6.

    Resuspend the cell in 100 µL of GTE buffer (seeNote 14).

     
  7. 7.

    Add 200 µL of 0.2N NaOH, 1% SDS, and invert several times

     
  8. 8.

    Add 150 µL of 5M potassium acetate, and invert 10 times.

     
  9. 9.

    Add 450 µL of 5M lithium chloride, and invert 10 times.

     
  10. 10.

    Put on ice for 5 min, and centrifuge at the full speed for 5 min.

     
  11. 11.

    Transfer supernatant to a new Eppendorf tube.

     
  12. 12.

    Add 600 µL of isopropanol.

     
  13. 13.

    Put on dry me for 5 min, and centrifuge at the full speed for 5 min.

     
  14. 14.

    Rinse the pellets with 75% ethanol, and air-dry.

     
  15. 15.

    Dissolve the pellets in 190 µL of H2O

     
  16. 16.

    Add 10 µL of RNase A (100 µg/µL).

     
  17. 17.

    Incubate at 37°C for 30 min.

     
  18. 18.

    Add 200 µL of phenol/CIAA, and vortex well.

     
  19. 19.

    Centrifuge at the full speed for 5 min to separate phases and save the aqueous phase for the next step.

     
  20. 20.

    Add 1 µL of glycogen (10 µg/µL), 20 µL of 3M sodium acetate, and 450 µL of ethanol, and put on dry ice for 20 min.

     
  21. 21.

    Spin at the full speed for 15 min, rinse the pellet with 1 mL of 75% ethanol, and air-dry.

     
  22. 22.

    Dissolve in 50 µL of H2O. Store at −20°C.

     
  23. 23.

    Using 2 µL of above DNA solution, and check the DNA concentration by running on the 1% agarose/TAE gel. At least 200 ng of DNA are needed in one transfection procedure.

     
  24. 24.

    Prepare pcDL-SRα-hRAR(5′)-Tac(3′) and pcDL-SRα-hG-CSF(5′)-Tac(3′) DNA using the same procedure as described in steps 423.

     

3.2.2 Transfection into COS-7 Cells

  1. 1.

    Mix 500 ng of pcDL-SRα-hRAR(5′)-Tac(3′) and H2O to 25 µL, and assign it to “Negative Control.” Mix 10 ng of pcDL-SRα-hG-CSF(5′)Tac(3′), 490 ng of pcDL-SRα-hRAR(5′)-Tac(3′), and H2O to 25 µL, and assign it to “Positive Control.”

     
  2. 2.

    Harvest exponentially growing COS-7 cells by trypsinization and replate six-well culture plate 7 × 104 cells/well 24 h before transfection. Add 2 mL of DMEM(+), and incubate at 37°C in a humidified incubator in an atmosphere of 5% CO2.

     
  3. 3.

    Remove the medium from the cells by aspiration, and wash twice with 2 mL of DMEM(−).

     
  4. 4.

    Add 605 µL of the DNA/DEAE-dextran solution consisting of 532 µL of DMEM(−), 30 µL of 1M Tris-HCl (pH 7.4), 12 µL of DEAE-dextran (10 mg/mL), 6 µL of chloroquine (10 mM), and 25 µL of plasmid solution prepared in Section 3.2.1.

     
  5. 5.

    Return the cells to the incubator, and incubate for 3 h.

     
  6. 6.

    Remove the solution, add 1 mL of 10% (v/v) DMSO, and set at room temperature for 2 min.

     
  7. 7.

    Remove the solution, and wash with 1.5 mL of DMEM(−) once and with 1.5 mL of DMEM(+) once.

     
  8. 8.

    Add 2 mL of DMEM(+), return to the incubator, and incubate for 44–72 h.

     

3.2.3 Cell Surface immunostaining with Antihuman CD25

  1. 1.

    Mix 50 µL of FITC-conjugated antihuman CD25 and 950 µL of PBS(−) supplemented with 1% (v/v) FCS. Set on ice (seeNote 15).

     
  2. 2.

    Remove the DMEM(+) from the COS-7 cells, and wash with 2 mL of PBS(−) twice.

     
  3. 3.

    Add 1 mL of 0.02% (w/v) EDTA.

     
  4. 4.

    Scrape off the COS-7 cells with cell scraper, suspend the cells with a pipetman p1000, and transfer the cells into Eppendorf tubes.

     
  5. 5.

    Spin at 8500g for 5 s, and remove the supernatant.

     
  6. 6.

    Add 20 µL of diluted FITC-conjugated antihuman CD25 prepared in step 1, and suspend well.

     
  7. 7.

    Set on ice for 20 min. Shield from light.

     
  8. 8.

    Tap the tube, add 800 µL of PBS(−) supplemented with 1% (v/v) FCS, spin at 8500g for 5 s, and remove the supernatant.

     
  9. 9.

    Tap the tube, add 800 µL of PBS(−) supplemented with 1% (v/v) FCS, suspend the cells with a pipetman p1000, spin at 8500g for 5 s, and remove the supernatant.

     
  10. 10.

    Resuspend the COS-7 cells in 6–10 µL of PBS(−) supplemented with 1% (v/v) FCS.

     
  11. 11.

    Observe with the fluorescent microscope.

     
  12. 12.

    In COS-7 cells transfected with “Positive Control” DNA, margins of 1 in 20–500 cells are glittering intensely. In other words, more than 10 surface staining positive cells are detected in 1 µL of “Positive Control” DNA transfected cell suspension prepared in Section 3.2.2., step 1. Only pools, in which COS-7 cells are transfected and that show surface fluorescence as strong as the cells transfected with the positive control plasmid should be judged as “positive.” For unknown reasons, a limited number of weak surface-stained cells can be sometimes observed in cells transfected with “Negative Control” DNA. Therefore, comparison in intensity and numbers with controls is needed to determine if a pool or clone is positive or negative. Typically, 2–8 positive clones are trapped in 1000 clones (20 pools) (seeNote 5).

     

3.2.4 Making Smaller Pools and identifying Single Positive Clones

  1. 1.

    Pick up seven individual colonies in a row or a line on the pool plate, and inoculate in 2 mL of terrific broth, containing 100 µg/mL of ampicillin. Fourteen sets of smaller pool consisting seven colonies should be prepared for one positive pool.

     
  2. 2.

    Prepare DNA from 14 sets of 2-mL cultures for one positive pool as described in Section 3.2.1., steps 4–22.

     
  3. 3.

    Transfect COS-7 cells with smaller pools as described in Section 3.2.2., and stain the cells with FITC-conjugated antihuman CD25 as described in Section 3.2.3.

     
  4. 4.

    One single positive clone can be obtained by determining which one in the smaller pool of seven rows and which one in the smaller pool of seven lines is positive (seeNote 16).

     
  5. 5.

    Prepare DNA of one clone assumed to be positive, transfect COS-7 cells with this DNA, and stain the transfected COS-7 cells with anti-Tac.

     

3.3 Analysis

3.3.1 Hydropathy Analysis and Database Search

  1. 1.

    Determine the base sequence of positive clones in both directions, using SRA primer and Tac primer (seeNote 7).

     
  2. 2.

    Make sure there is a start codon followed by an open reading frame fused with Tac (3′). Check if the base-sequence near the ATG fits with Kozak’s rule (12), or there are one or more m-frame stop codons upstream of the start codon. Either is enough (seeNote 17).

     
  3. 3.

    Draw the hydropathy profiles of deduced amino acid sequences of positive clones, compare the shape of hydropathy profiles with that of authentic N-terminal signal sequences, and make sure the putative N-terminal regions are as hydrophobic as authentic ones (seeNote 17) (13,14).

     
  4. 4.

    Calculate to make sure there is a reasonable cleavage site by signal peptidase by von Heme’s method using Gene Works program (seeNote 17) (14).

     
  5. 5.

    Compare the sequence information with the databases in both DNA and protein levels for homology using searching programs, such as BLAST or FASTA.

     
  6. 6.

    Using obtained cDNA fragments as probes, you may check the RNA expression and may screen an oligo(dT)-primed cDNA library to obtain full-length clones. Since a full coding region can never be trapped by Signal Sequence Trap, the screening for a full-length clone is always needed.

     
  7. 7.

    Draw the hydropathy profiles of deduced amino acid sequences of full-length clones, and make sure the N-terminal regions are hydrophobic (seeNote 18).

     
  8. 8.

    Compare the full-length sequence information with database in both DNA and protein levels for homology.

     
  9. 9.

    Check whether the deduced proteins have ER or GA retention signals (15).

     

4 Notes

  1. 1.
    The 300–500 bp fraction should be tested first for two reasons:
    1. a.

      Longer cDNAs, which contain the whole coding regions, do not generate fusion proteins because of the appearance of the stop codons.

       
    2. b.

      In fractions shorter than 200 bp, too many artifacts appear in our experience.

       
     
  2. 2.

    The sib-screening method described here is faster than FACS sorting followed by hirt fraction plasmid recovery.

     
  3. 3.

    cDNA construction should be started with 200 ng-5 µg of polyA+ RNA. Contamination with rRNA will result in increasing the clone number you have to screen.

     
  4. 4.

    To enrich 5′-end of cDNA, <20 ng of random hexamer primer should be used for 1 µg of poly A+ RNA.

     
  5. 5.

    Probability of the positive clones is typically 1/100–1/500 but can be 1/80–1/1500, depending on quality of polyA+ RNA, random hexamer/polyA+ RNA ratio, elongation of first-strand cDNA synthesis, size fraction, and cell source.

     
  6. 6.

    The elongation of cDNA can be monitored by adding [α-32P] dCTP and applying it on an alkaline agarose gel.

     
  7. 7.

    If dC-tailing is too long, there will be some difficulties in determination of base sequences, because poly(dG) longer than 25 bases prevents sequencing reaction 1/2X reverse transcription buffer, which contains as low as 1.5 mM MgCl2, tends to give better results than tailing buffer containing cacodylate (16), although cacodylate has been recommended for use for a long time (10,17,18). If there is still trouble in base sequencing, another sequencing primer, SLG9 (20-mer) 5′-GACTAACTGACGGGGGGGGG can be tried.

     
  8. 8.

    The combination of dA-tailing and poly(dT) primer can be used instead of the combinations of dG-tailing and poly(dC) primer. Poly(dT) does not prevent the sequencing reaction so severely as poly(dG).

     
  9. 9.
    Cutting at the middle of coding region of cDNA by shearing with ultrasound sonication may bring more efficient fusion protein generation, although sometimes, recovery of DNA after sonication is rather poor. Sonication steps can be added as follows:
    1. a.

      Add H2O to the double-stranded cDNA prepared in Section 3.1.2., step 14 to the final volume of 400 µL.

       
    2. b.

      Sonicate 5–10 times for 30 s at setting 5, continuous output, 100% duty cycle in a 15-mL disposable tube, chilling with ice-cold water, using SONICATOR (HEAT-SYSTEM-ULTRASONICS, Farmingdale, NY). Before shearing cDNA, you should practice sonication using such DNA fragments as λ-phage DNA digested with HindIII, and determine the settings and time.

       
    3. c.

      Transfer to an Eppendorf tube, do phenol/CIAA extraction, ethanol precipitation, rinse, and dissolve DNA in 7 µL of H2O.

       
    4. d.

      Add 1 µL of 10X second-strand buffer, 1 µL of 2 mM dNTP, vortex, and quick spin.

       
    5. e.

      Add 1 µL of T4 DNA polymerase (3 U/µL), stir gently, and quick spin.

       
    6. f.

      Incubate at 37°C for 10 min, then vortex violently, and spin.

       
    7. g.

      Incubate at 75°C for 15 min, quickly chill on ice, and go to Section 3.1.4.

       
     
  10. 10.

    In Section 3.1.3., steps 19–24, DNA recovery method using Geneclean II kit is recommended because the loss of around 300 bp DNA fragments was minimum in our hands.

     
  11. 11.

    Instead of Taq DNA polymerase, PFU DNA polymerase (Stratagene) or Deep Vent DNA Polymerase (New England Biolab), may be useful to avoid mutation during PCR.

     
  12. 12.

    When smearing of DNA fragments is observed in the 1/1000 diluted reaction, even if it is very faint, there will be little chance to trap exactly the same clones generated by PCR.

     
  13. 13.

    For vector preparation, negative control plasmid, pcDL-SRα-hRAR(5′)-Tac(3′) should be used.

     
  14. 14.

    The Wizard miniprep Kit (Promega, Madison, WI) may be used, instead of steps 6–22 of Section 3.2.1.

     
  15. 15.

    The optimal concentrations of antibodies should be titrated before use. Alternatively, the combination of anti-Tac monoclonal antibodies (MAb) (anti-Tac ascitis can be obtained from T. A. Waldman (NIH Bethesda) or T. Uchiyama (Kyoto University, Kyoto, Japan) and FITC-conjugated AffinPure goat antimouse IgG (H + L) (1 mg/mL) (Jackson ImmunoReseach Laboratories, West Grove, PA) can be used.

     
  16. 16.

    In 10% of positive pools obtained in the first screening, no positive clones appear in the secondary screening for unknown reasons. Sometimes more than one clone appears from one positive pool in the secondary screening.

     
  17. 17.

    In the fraction (300–500 bp), 15–30% of the anti-Tac surface-stained positive clones do not match the three criteria described in Section 3.3.1.) steps 2–4. The ratio of artificial clones depends on the stringency of the judgment of anti-Tac staining (4).

     
  18. 18.

    Results of computer homology search or cloning of fill-length cDNA shows that 10–30% of the clones which match the three criteria (Section 3.3.1., steps 2–4) do not encode N-terminal, but the middle of the coding regions. Some of them code putative transmembrane regions. The ratio depends on the stringency of the judgment of the existence of N-terminal signal sequence in Section 3.3.1., step 3 (4).

     

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Copyright information

© Humana Press Inc., Totowa, NJ 1997

Authors and Affiliations

  • Kei Tashiro
    • 1
  • Toru Nakano
    • 1
  • Tasuku Honjo
    • 1
  1. 1.Department of Medical Chemistry, Faculty of MedicineKyoto UniversityKyotoJapan

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