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. 1993 Nov 1;90(21):10330–10334. doi: 10.1073/pnas.90.21.10330

Formation of functional peptide complexes of class II major histocompatibility complex proteins from subunits produced in Escherichia coli.

J D Altman 1, P A Reay 1, M M Davis 1
PMCID: PMC47768  PMID: 8234294

Abstract

Class II major histocompatibility complex molecules play a major role in the immune response by binding peptide fragments of exogenous antigens and displaying them on the surfaces of antigen-presenting cells, where they can be recognized by T cells. To facilitate structural and functional studies of these molecules, we have produced truncated alpha and beta chains of the murine class II molecule I-Ek in Escherichia coli (Ec-I-Ek) and have developed conditions to fold them in the presence of specific peptides with yields of complex approaching 2%. Reconstitution is specific since only unlabeled peptide known to bind I-Ek compete with biotinylated peptide, as assessed by ELISA. Complexes of the refolded heterodimer (Ec-I-Ek) with either of two different peptide antigens remain associated during nonreducing SDS/PAGE. Immobilized Ec-I-Ek-peptide complexes stimulate lymphokine production by three T-cell clones in an antigen-specific manner with a dose-response relation comparable to previously described soluble I-Ek molecules produced in CHO cells. These results demonstrate that folding of Ek alpha and Ek beta polypeptides does not require any other protein to produce the biologically relevant conformation and that carbohydrate modification of this class II molecule is not necessary for alpha beta T-cell recognition.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altman J. D., Henner D., Nilsson B., Anderson S., Kuntz I. D. Intracellular expression of BPTI fusion proteins and single column cleavage/affinity purification by chymotrypsin. Protein Eng. 1991 Jun;4(5):593–600. doi: 10.1093/protein/4.5.593. [DOI] [PubMed] [Google Scholar]
  2. Anfinsen C. B. Principles that govern the folding of protein chains. Science. 1973 Jul 20;181(4096):223–230. doi: 10.1126/science.181.4096.223. [DOI] [PubMed] [Google Scholar]
  3. Bjorkman P. J., Parham P. Structure, function, and diversity of class I major histocompatibility complex molecules. Annu Rev Biochem. 1990;59:253–288. doi: 10.1146/annurev.bi.59.070190.001345. [DOI] [PubMed] [Google Scholar]
  4. Bjorkman P. J., Saper M. A., Samraoui B., Bennett W. S., Strominger J. L., Wiley D. C. Structure of the human class I histocompatibility antigen, HLA-A2. Nature. 1987 Oct 8;329(6139):506–512. doi: 10.1038/329506a0. [DOI] [PubMed] [Google Scholar]
  5. Braunstein N. S., Germain R. N., Loney K., Berkowitz N. Structurally interdependent and independent regions of allelic polymorphism in class II MHC molecules. Implications for Ia function and evolution. J Immunol. 1990 Sep 15;145(6):1635–1645. [PubMed] [Google Scholar]
  6. Brown J. H., Jardetzky T. S., Gorga J. C., Stern L. J., Urban R. G., Strominger J. L., Wiley D. C. Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature. 1993 Jul 1;364(6432):33–39. doi: 10.1038/364033a0. [DOI] [PubMed] [Google Scholar]
  7. Buchner J., Rudolph R. Renaturation, purification and characterization of recombinant Fab-fragments produced in Escherichia coli. Biotechnology (N Y) 1991 Feb;9(2):157–162. doi: 10.1038/nbt0291-157. [DOI] [PubMed] [Google Scholar]
  8. Dornmair K., McConnell H. M. Refolding and reassembly of separate alpha and beta chains of class II molecules of the major histocompatibility complex leads to increased peptide-binding capacity. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4134–4138. doi: 10.1073/pnas.87.11.4134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Driscoll P. C., Altman J. D., Boniface J. J., Sakaguchi K., Reay P. A., Omichinski J. G., Appella E., Davis M. M. Two-dimensional nuclear magnetic resonance analysis of a labeled peptide bound to a class II major histocompatibility complex molecule. J Mol Biol. 1993 Jul 20;232(2):342–350. doi: 10.1006/jmbi.1993.1394. [DOI] [PubMed] [Google Scholar]
  10. Garboczi D. N., Hung D. T., Wiley D. C. HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3429–3433. doi: 10.1073/pnas.89.8.3429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jackson M. R., Song E. S., Yang Y., Peterson P. A. Empty and peptide-containing conformers of class I major histocompatibility complex molecules expressed in Drosophila melanogaster cells. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):12117–12121. doi: 10.1073/pnas.89.24.12117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jensen P. E. Enhanced binding of peptide antigen to purified class II major histocompatibility glycoproteins at acidic pH. J Exp Med. 1991 Nov 1;174(5):1111–1120. doi: 10.1084/jem.174.5.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jorgensen J. L., Reay P. A., Ehrich E. W., Davis M. M. Molecular components of T-cell recognition. Annu Rev Immunol. 1992;10:835–873. doi: 10.1146/annurev.iy.10.040192.004155. [DOI] [PubMed] [Google Scholar]
  14. Ljunggren H. G., Stam N. J., Ohlén C., Neefjes J. J., Höglund P., Heemels M. T., Bastin J., Schumacher T. N., Townsend A., Kärre K. Empty MHC class I molecules come out in the cold. Nature. 1990 Aug 2;346(6283):476–480. doi: 10.1038/346476a0. [DOI] [PubMed] [Google Scholar]
  15. Madden D. R., Gorga J. C., Strominger J. L., Wiley D. C. The three-dimensional structure of HLA-B27 at 2.1 A resolution suggests a general mechanism for tight peptide binding to MHC. Cell. 1992 Sep 18;70(6):1035–1048. doi: 10.1016/0092-8674(92)90252-8. [DOI] [PubMed] [Google Scholar]
  16. Matsui K., Boniface J. J., Reay P. A., Schild H., Fazekas de St Groth B., Davis M. M. Low affinity interaction of peptide-MHC complexes with T cell receptors. Science. 1991 Dec 20;254(5039):1788–1791. doi: 10.1126/science.1763329. [DOI] [PubMed] [Google Scholar]
  17. Matsumura M., Fremont D. H., Peterson P. A., Wilson I. A. Emerging principles for the recognition of peptide antigens by MHC class I molecules. Science. 1992 Aug 14;257(5072):927–934. doi: 10.1126/science.1323878. [DOI] [PubMed] [Google Scholar]
  18. Reay P. A., Wettstein D. A., Davis M. M. pH dependence and exchange of high and low responder peptides binding to a class II MHC molecule. EMBO J. 1992 Aug;11(8):2829–2839. doi: 10.1002/j.1460-2075.1992.tb05350.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sadegh-Nasseri S., Germain R. N. How MHC class II molecules work: peptide-dependent completion of protein folding. Immunol Today. 1992 Feb;13(2):43–46. doi: 10.1016/0167-5699(92)90131-P. [DOI] [PubMed] [Google Scholar]
  20. Schrader J. W., Clark-Lewis I., Crapper R. M., Wong G. W. P-cell stimulating factor: characterization, action on multiple lineages of bone-marrow-derived cells and role in oncogenesis. Immunol Rev. 1983;76:79–104. doi: 10.1111/j.1600-065x.1983.tb01098.x. [DOI] [PubMed] [Google Scholar]
  21. Silver M. L., Parker K. C., Wiley D. C. Reconstitution by MHC-restricted peptides of HLA-A2 heavy chain with beta 2-microglobulin, in vitro. Nature. 1991 Apr 18;350(6319):619–622. doi: 10.1038/350619a0. [DOI] [PubMed] [Google Scholar]
  22. Springer T. A., Kaufman J. F., Siddoway L. A., Mann D. L., Strominger J. L. Purification of HLA-linked B lymphocyte alloantigens in immunologically active form by preparative sodium dodecyl sulfate-gel electrophoresis and studies on their subunit association. J Biol Chem. 1977 Sep 10;252(17):6201–6207. [PubMed] [Google Scholar]
  23. Stern L. J., Wiley D. C. The human class II MHC protein HLA-DR1 assembles as empty alpha beta heterodimers in the absence of antigenic peptide. Cell. 1992 Feb 7;68(3):465–477. doi: 10.1016/0092-8674(92)90184-e. [DOI] [PubMed] [Google Scholar]
  24. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  25. Tandon S., Horowitz P. Detergent-assisted refolding of guanidinium chloride-denatured rhodanese. The effect of lauryl maltoside. J Biol Chem. 1986 Nov 25;261(33):15615–15618. [PubMed] [Google Scholar]
  26. Watson J. Continuous proliferation of murine antigen-specific helper T lymphocytes in culture. J Exp Med. 1979 Dec 1;150(6):1510–1519. doi: 10.1084/jem.150.6.1510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wettstein D. A., Boniface J. J., Reay P. A., Schild H., Davis M. M. Expression of a class II major histocompatibility complex (MHC) heterodimer in a lipid-linked form with enhanced peptide/soluble MHC complex formation at low pH. J Exp Med. 1991 Jul 1;174(1):219–228. doi: 10.1084/jem.174.1.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zhang W., Young A. C., Imarai M., Nathenson S. G., Sacchettini J. C. Crystal structure of the major histocompatibility complex class I H-2Kb molecule containing a single viral peptide: implications for peptide binding and T-cell receptor recognition. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8403–8407. doi: 10.1073/pnas.89.17.8403. [DOI] [PMC free article] [PubMed] [Google Scholar]

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