Preparation of Stable Single‐Chain Trimers Engineered with Peptide, β2 Microglobulin, and MHC Heavy Chain

Ted Hansen1, Y.Y. Lawrence Yu1, Daved H. Fremont1

1 Washington University School of Medicine, St. Louis, Missouri
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 17.5
DOI:  10.1002/0471142735.im1705s87
Online Posting Date:  November, 2009
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This unit describes a method for constructing a class I MHC molecule with a bound peptide as a single polypeptide chain, termed SCT, for single chain trimer. The component organization of the SCT appears to be widely applicable to different mouse or human MHC class I isotypes bound by different antigenic peptides. The enhanced peptide occupancy afforded by the SCT format makes these molecules effective reagents as DNA vaccines, multimeric staining reagents to enumerate CD8 T cells, and probes of lymphocyte biology. Curr. Protoc. Immunol. 87:17.5.1‐17.5.17. © 2009 by John Wiley & Sons, Inc.

Keywords: vaccines; tetramers; lymphocyte development; recombinant MHC/peptide complexes

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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Construction of SCT for Mammalian Expression
  • Basic Protocol 2: Production of SCT Monomer and Tetramer
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Construction of SCT for Mammalian Expression

  • β2mb cDNA and H‐2Kb cDNA
  • Expand High Fidelity PCR kit (Roche)
  • Sense oligo encoding a NotI site plus the start of the β2m signal peptide. (Oligo 1 in Table 17.5.2)
  • Antisense oligo encoding the end of the β2m coding sequence plus G4SG4SG linker (wherein the last two residues SG are encoded by the BspEI site or TCCGGA; Oligo 2 in Table 17.5.2)
  • Sense oligo encoding a SG4SG4S linker (wherein the first two residues SG are encoded by BspEI) plus the start of the Kb mature peptide (Oligo 3 in Table 17.5.2)
  • Antisense oligo encoding the end of the Kb coding sequence plus STOP codon plus a BamHI site (Oligo 4 in Table 17.5.2)
  • QIAquick PCR Purification Kit (Qiagen)
  • Restriction enzymes: NotI, BspEI, BamHI, DpnI, and NheI (New England Biolabs)
  • pIRESneo plasmid (BD Clontech)
  • Calf intestinal phosphatase (CIP; New England Biolabs)
  • Rapid DNA Ligation Kit (Roche)
  • XL‐2 Blue Ultracompetent E. coli (Stratagene)
  • LB agar plates (see recipe) containing 100 µg/ml ampicillin
  • LB liquid medium (see recipe) containing 100 µg/ml ampicillin
  • QIAprep Spin Miniprep Kit (Qiagen)
  • BigDye Terminator mix v. 3.1 (Perkin‐Elmer)
  • Antisense oligo that encodes the end of the β2m signal peptide plus SIINFEKL (ovalbumin derived Kb epitope) plus G3AS, wherein the last two residues AS are encoded by the NheI site or GCTAGC (Oligo 5 in Table 17.5.2)
  • Sense oligo that encodes ASG4SG4S plus the start of the β2m mature portion wherein the first two residues AS are encoded by the NheI site (Oligo 6 in Table 17.5.2)
  • QuikChange mutagenesis kit (Stratagene)
  • Mouse L cells (e.g., ATCC CRL‐2648) or other cell line easy to tranfect and transduce, e.g., mouse 3T3 (ATCC CCL‐163) cells, human 293 (ATCC CRL‐1573), or HeLa cells)
  • Complete RPMI medium/10% FBS ( appendix 2A)
  • Opti‐MEM serum‐free medium (Invitrogen)
  • Fugene transfection reagent (Roche)
  • Complete RPMI medium/10% FBS ( appendix 2A) supplemented with 1 mg/ml G418
  • Thermal cycler
  • 6‐well tissue culture plates
  • 5‐ml polystyrene tubes
  • Additional reagents and equipment for PCR (unit 10.20), agarose gel electrophoresis (unit 10.4& ), transformation of E. coli ( appendix 3N), and restriction mapping (Bloch, )
    Table 7.5.2   MaterialsOligonucleotides Used for PCR in This Unit

    Oligo no. Sequence (5′‐3′)
    1 tctgcggccgcgtcgacggaatttcgactctagaagcatggctcgctcggtgacc
    2 gcaacttccggaaccgcctccaccggaaccaccaccgcccatgtctcgatcccagtag
    3 gcaacttccggaggtggtggatccggtggtggtggtagtggcccacactcgctgagg
    4 gatggatcctcacgctttacaatctgggag
    5 gaggctagcacctcctccaagtttttcgaaattaatgatactagcatacaaaccggtcagtg
    6 ggtgctagcggtggtggtggtagcggaggtggaggcagcatccagaaaacccctcaaatt

Basic Protocol 2: Production of SCT Monomer and Tetramer

  • SCT DNA in pIRESneo vector ( protocol 1)
  • Expand High Fidelity PCR Kit (Roche)
  • pET21a plasmid (Novagen)
  • BL21‐CodonPlus (DE3)‐RIL competent E. coli cells (Stratagene)
  • LB liquid medium (see recipe) containing 100 µg/ml carbenicillin
  • Sodium deoxycholate
  • Guanidinium⋅HCl–based solubilization buffer: 6 M guanidine hydrochloride/20 mM Tris⋅Cl, pH 8.0; add 2‐mercaptoethanol to 20 mM final just before use
  • Size exclusion chromatography buffer: 20 mM HEPES, pH 7.5, 150 mM NaCl, and 0.01% NaN 3
  • Ion‐exchange buffer: 25 mM Tris⋅Cl, pH 7.5 ( appendix 2A)
  • NaCl
  • BirA ligase (Avidity;
  • PE‐labeled streptavidin (BD Pharmingen)
  • Thermal cycler
  • 250‐ml Erlenmeyer flask
  • 37°C incubator with shaker
  • Millipore Stirred Ultrafiltration Cell Model 8400 with 30‐KDa MWCO membrane
  • Millex‐GV low‐protein‐binding syringe filter (Millipore)
  • ÄKTA FPLC system (GE Healthcare Life Sciences) including:
    • Superdex 75 size‐exclusion column
    • Superdex 200 size‐exclusion column
    • Mono Q 5/50 GL ion‐exchange column
  • Amicon Ultra centrifugal filter unit (Millipore) with 30‐kDa MWCO membrane
  • Avanti J‐26 XP Series High‐Performance Centrifuge with JLA 8.1000 and JLA 16.250 rotors (Beckman Coulter)
  • 0.2‐µm bottle‐top filtration unit with 0.2‐µm pore‐size PES membrane (Nalgene)
  • Spectrophotometer
  • Additional reagents and equipment for cloning SCT into a plasmid ( protocol 1), oxidative refolding of protein (unit 17.3), SDS‐PAGE and staining of proteins in gels (unit 8.4), and immunoblotting (unit 8.10)
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Literature Cited

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