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Production of Stabilized Antibody Fragments in the E. coli Bacterial Cytoplasm and in Transiently Transfected Mammalian Cells

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Human Monoclonal Antibodies

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1904))

Abstract

Monoclonal antibodies (mAbs) are currently the fastest growing class of therapeutic proteins. Parallel to full-length IgG format the development of recombinant technologies provided the production of smaller recombinant antibody variants. The single-chain variable fragment (scFv) antibody is a minimal form of functional antibody comprised of the variable domains of immunoglobulin light and heavy chains connected by a flexible linker. In most cases, scFvs are expressed in the periplasm bacterium E. coli. The production of soluble scFvs is more effective in quantity, however, under the reducing conditions of the E. coli bacterial cytoplasm it is inefficient because of the inability of the disulfide bonds to form. Hence, scFvs are either secreted to the periplasm as soluble proteins or expressed in the cytoplasm as insoluble inclusion bodies and recovered by refolding. The cytoplasmic expression of scFvs as a C-terminal fusion to maltose-binding protein (MBP) provided the high-level production of stable, soluble, and functional fusion protein. The below protocol provides the detailed description of MBP-scFv production in E. coli utilizing two expression systems: pMALc-TNN and pMALc-NHNN. Although the MBP tag does not disrupt the most of antibody activities, the MBP-TNN-scFv product can be cleaved by Tobacco Etch Virus (TEV) protease in order to obtain untagged scFv.

The second protocol is for efficient production of Fab antibody fragments as MBP fusion proteins secreted by transiently transfected mammalian cells. While transient transfection is a fast and effective way of obtaining several mgs of antibody for initial screening and validation of antibodies, some antibody sequences express poorly or not at all. For such antibodies, fusion to MBP provides an effective approach for solving the expression problem.

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Author notes

  1. Racheli Birnboim-Perach and Yehudit Grinberg contributed equally to this work.

Authors and Affiliations

  1. School of Molecular Cell Biology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv, Israel

    Racheli Birnboim-Perach, Yehudit Grinberg, Lilach Vaks, Limor Nahary & Itai Benhar

Authors
  1. Racheli Birnboim-Perach
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  2. Yehudit Grinberg
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  3. Lilach Vaks
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  4. Limor Nahary
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  5. Itai Benhar
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Corresponding author

Correspondence to Itai Benhar .

Editor information

Editors and Affiliations

  1. Department of Pathology, The Hebrew University, Hadassah Medical School, Jerusalem, Israel

    Michael Steinitz

Appendix: Sequences of Plasmids (Complete Sequences Are Available from the Authors Upon Request)

Appendix: Sequences of Plasmids (Complete Sequences Are Available from the Authors Upon Request)

The sequence of pMALc2 from NEB is available at: https://international.neb.com/-/media/nebus/page-images/tools-and-resources/interactive-tools/dna-sequences-and-maps/text-documents/pmalc2gbk.txt?la=en

  1. 1.

    pMALc-TNN-scFv35 (the cloned scFv is an anti HCV NS3 protease scFv that was earlier described [25]. To create pMALc-TNN-scFv35, insert the following sequence between coordinates 2676 to 2727 of pMALc2. In the sequence below, the scFv (including the C-terminal His tag and Myc tag) is cloned between positions 31 to 847.

    tccGAGaacCTCtacTTCcagTccatggccGAGGTCCAGCTGCAGCAATCTGGAGCAGAGCTTGTGAGGTCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACTACTATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAGAATGGTGATACTGAATACACTCAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCCCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAATTACTACGGATTACTACTTTGACTACTGGGGCCAAGGCACCACGCTCACCGTCTCCTCGggaggtggtggatccggcggtggcggttctggtggaggtggatctGATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCTCTCACGTTCGGTGCTGGGACCAAACTGGAGATCAAACGGgcggccgcACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATcTCAGAAGAGGATCTGAATggggccgcaT

  2. 2.

    To create pMALc-NHNN-scFv35 (the cloned scFv is an anti HCV NS3 protease scFv that was described in [25]. To create pMALc-NHNN-scFv35, insert the following sequence between coordinates 1524 to 2723 of pMALc2. In the sequence below, there is a His tag before the MBP ORF is between positions 7 to 24. The scFv (including the C-terminal His tag and Myc tag) is cloned between positions 1186 to 1950.

    catatgCACCATCACCATCACCATtccggcAAAACTGAAGAAGGTAAACTGGTAATCTGGATTAACGGCGATAAAGGCTATAACGGTCTCGCTGAAGTCGGTAAGAAATTCGAGAAAGATACCGGAATTAAAGTCACCGTTGAGCATCCGGATAAACTGGAAGAGAAATTCCCACAGGTTGCGGCAACTGGCGATGGCCCTGACATTATCTTCTGGGCACACGACCGCTTTGGTGGCTACGCTCAATCTGGCCTGTTGGCTGAAATCACCCCGGACAAAGCGTTCCAGGACAAGCTGTATCCGTTTACCTGGGATGCCGTACGTTACAACGGCAAGCTGATTGCTTACCCGATCGCTGTTGAAGCGTTATCGCTGATTTATAACAAAGATCTGCTGCCGAACCCGCCAAAAACCTGGGAAGAGATCCCGGCGCTGGATAAAGAACTGAAAGCGAAAGGTAAGAGCGCGCTGATGTTCAACCTGCAAGAACCGTACTTCACCTGGCCGCTGATTGCTGCTGACGGGGGTTATGCGTTCAAGTATGAAAACGGCAAGTACGACATTAAAGACGTGGGCGTGGATAACGCTGGCGCGAAAGCGGGTCTGACCTTCCTGGTTGACCTGATTAAAAACAAACACATGAATGCAGACACCGATTACTCCATCGCAGAAGCTGCCTTTAATAAAGGCGAAACAGCGATGACCATCAACGGCCCGTGGGCATGGTCCAACATCGACACCAGCAAAGTGAATTATGGTGTAACGGTACTGCCGACCTTCAAGGGTCAACCATCCAAACCGTTCGTTGGCGTGCTGAGCGCAGGTATTAACGCCGCCAGTCCGAACAAAGAGCTGGCGAAAGAGTTCCTCGAAAACTATCTGCTGACTGATGAAGGTCTGGAAGCGGTTAATAAAGACAAACCGCTGGGTGCCGTAGCGCTGAAGTCTTACGAGGAAGAGTTGGCGAAAGATCCACGTATTGCCGCCAcTatggAAAACGCCCAGAAAGGTGAAATCATGCCGAACATCCCGCAGATGTCCGCTTTCTGGTATGCCGTGCGTACTGCGGTGATCAACGCCGCCAGCGGTCGTCAGACTGTCGATGAAGCCCTGAAAGACGCGCAGACTAATTCGAGCTCggtaccgtcctctctcgtgatcgagggtaggcctgaattcagtaccatggccGAGGTCCAGCTGCAGCAATCTGGAGCAGAGCTTGTGAGGTCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACTACTATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAGAATGGTGATACTGAATACACTCAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCCCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAATTACTACGGATTACTACTTTGACTACTGGGGCCAAGGCACCACGCTCACCGTCTCCTCGggaggtggtggatccggcggtggcggttctggtggaggtggatctGATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCTCTCACGTTCGGTGCTGGGACCAAACTGGAGATCAAACGGgcggccgcagactacaaggact

    The sequence of plasmid pcDNA3.1 is available at: https://www.ncbi.nlm.nih.gov/nuccore/EF550208.1

  3. 3.

    To create pcDNA3.4-Fd-His (an expression vector for an antibody Fd domain with a C-terminal His tag), insert the following sequence between coordinates 820 to 2912 of pcDNA3.1. (The resulting pcDNA3.4-Fd-His carries the cloned VH-CH1 is of the therapeutic monoclonal anti TNFα antibody Infliximab). In the sequence below, the secretion leader sequence ORF spans positions 147 to 203. The Fd (VH+CH1) (including a C-terminal His tag and stop codon) spans positions 204 to 911. In the sequence shown herein, the His tag and stop codon were inserted between the end of the human Gamma1 CH1 to the hinge region (spanning positions 882 to 908). Upon removal of the sequence spanning positions 882 to 911, a full human IgG1 heavy chain ORF will be restored.

    GTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGGACTCTAGAGGATCGAACCCTTGGATCTCTAGCGAATTCCCTCTAGACACAGACGCTCACCATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTGCTCAAAGGTGTCCAGTGTGAAGTGAAGCTTGAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCATTTTCAGTAACCACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGATCAAAATCTATTAATTCTGCAACACATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTGCTGTGTACCTGCAAATGACCGACTTAAGAACTGAAGACACTGGCGTTTATTACTGTTCCAGGAATTACTACGGTAGTACCTACGACTACTGGGGCCAAGGCACCACTCTCACAGTGTCCTCCgctAGCaccaagggcccatcggtcTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTggcggctcccatcaccatcaccatcacTGAGAGCCCAAATCTTGtGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAaCCATCtCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAcGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAAtgAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGACCTCGACAAGGGTTCGATCCCTACCGGTTAGTAATGAGTTTGATATCTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAAACGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCTGCCATAGCAGATCTGCGCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCC

  4. 4.

    To create pcDNA3.4-MBP-Fd-His insert the following sequence between positions 837 to 840 of pcDNA3.4-Fd-His. (the cloned VH-CH1 is of the therapeutic monoclonal anti TNFα antibody Infliximab). In the sequence below is the MBP ORF.

    AAAACTGAAGAAGGTAAACTGGTAATCTGGATTAACGGCGATAAAGGCTATAACGGTCTCGCTGAAGTCGGTAAGAAATTCGAGAAAGATACCGGAATTAAAGTCACCGTTGAGCATCCGGATAAACTGGAAGAGAAATTCCCACAGGTTGCGGCAACTGGCGATGGCCCTGACATTATCTTCTGGGCACACGACCGCTTTGGTGGCTACGCTCAATCTGGCCTGTTGGCTGAAATCACCCCGGACAAAGCGTTCCAGGACAAGCTGTATCCGTTTACCTGGGATGCCGTACGTTACAACGGCAAGCTGATTGCTTACCCGATCGCTGTTGAAGCGTTATCGCTGATTTATAACAAAGATCTGCTGCCGAACCCGCCAAAAACCTGGGAAGAGATCCCGGCGCTGGATAAAGAACTGAAAGCGAAAGGTAAGAGCGCGCTGATGTTCAACCTGCAAGAACCGTACTTCACCTGGCCGCTGATTGCTGCTGACGGGGGTTATGCGTTCAAGTATGAAAACGGCAAGTACGACATTAAAGACGTGGGCGTGGATAACGCTGGCGCGAAAGCGGGTCTGACCTTCCTGGTTGACCTGATTAAAAACAAACACATGAATGCAGACACCGATTACTCCATCGCAGAAGCTGCCTTTAATAAAGGCGAAACAGCGATGACCATCAACGGCCCGTGGGCATGGTCCAACATCGACACCAGCAAAGTGAATTATGGTGTAACGGTACTGCCGACCTTCAAGGGTCAACCATCCAAACCGTTCGTTGGCGTGCTGAGCGCAGGTATTAACGCCGCCAGTCCGAACAAAGAGCTGGCGAAAGAGTTCCTCGAAAACTATCTGCTGACTGATGAAGGTCTGGAAGCGGTTAATAAAGACAAACCGCTGGGTGCCGTAGCGCTGAAGTCTTACGAGGAAGAGTTGGCGAAAGATCCACGTATTGCCGCCAcTatggAAAACGCCCAGAAAGGTGAAATCATGCCGAACATCCCGCAGATGTCCGCTTTCTGGTATGCCGTGCGTACTGCGGTGATCAACGCCGCCAGCGGTCGTCAGACTGTCGATGAAGCCCTGAAAGACGCGCAGACTAATTCGAGCTCggtaccgtcctctctcgtgatcgagggtaggcctgaattcagtaccatggcc

  5. 5.

    To create pcDNA3.4-IFX-Kappa (an expression vector for a Kappa light chain. The cloned Kappa light chain is of the chimeric therapeutic monoclonal anti TNFα antibody Infliximab) insert the following sequence between position 819 to 2907 of pcDNA3.1 In the sequence below, the secretion leader sequence ORF spans positions 122 to 190. The Kappa light chain (mouse Vκ + human Cκ) spans positions 191 to 832.

    GTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGGACTCTAGAGGATCGAACCCTTAGGCAGGACCCAGCATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTACTGCTCTGGCTCCCAGGTGCCAGATGTGCCGACATCTTGCTGACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGTTCGTTGGCTCAAGCATCCACTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATGTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACACTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAAGAAAGTCATAGCTGGCCATTCACGTTCGGCTCGGGGACAAATTTGGAAGTAAAACGCACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAAGGGTTCGATCCCTACCGGTTAGTAATGAGTTTAAACTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGAAACGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCTGCCATAGCAGATCTGCG

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Birnboim-Perach, R., Grinberg, Y., Vaks, L., Nahary, L., Benhar, I. (2019). Production of Stabilized Antibody Fragments in the E. coli Bacterial Cytoplasm and in Transiently Transfected Mammalian Cells. In: Steinitz, M. (eds) Human Monoclonal Antibodies. Methods in Molecular Biology, vol 1904. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8958-4_23

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  • DOI: https://doi.org/10.1007/978-1-4939-8958-4_23

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8957-7

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