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closeIs this technology truly preferable?
Posted by Ton_Schumacher on 29 Feb 2008 at 14:06 GMT
I wish to congratulate the authors with the development of the process outlined here and applied to the production of in total 7 different MHC-ligand complexes.
It does seem however that the authors may underestimate the power of the UV-induced MHC exchange approach previously published by us to -well at least- some extent.
First, in a paper that was originally not referenced by the authors we performed a medium-throughput screen of the H5N1 avian flu genome using a set of 150 HLA-A2 multimers generated in 1 hr exchange reactions (1).
Second, in more recent work we have screened a series of some 200 peptides from melanoma-associated antigens for binding to the HLA-A3 allele (2).
Third, in work recently published by Ploegh and colleagues, an MHC exchange screen of 2,000 epitopes was performed to identify H2Db- and Kb-restricted Chlamydia epitopes in a murine infection model (3).
Fourth, in unpublished work we have assessed the binding of >21,000 potential ligands to HLA-A2 using this technology.
Thus, with UV-induced MHC ligand exchange established for 7 alleles (1-3) and applied to some 23,000 different potential MHC-ligand complexes we would consider the approach rather well established.
As a final technical issue, it is worth pointing out that the disulfide-containing MHC heavy chains used by the authors are obtained in a 4-step (sic) purification with low yield (Ref 11 in the paper), something that is impossible to glean from the current M&M but does seem of some relevance.
1). M. Toebes, M. Coccoris, A. Bins, B. Rodenko, R. Gomez, N.J. Nieuwkoop, W. van de Kasteele, G.F. Rimmelzwaan, J.B.A.G. Haanen, H. Ovaa and T.N.M. Schumacher. Design and use of conditional MHC class I ligands. Nat. Med. 12:246-251 (2006).
2). A.H. Bakker, M. Toebes, C. Linnemann, B. Rodenko, S. Reker-Hadrup, W.J.E. van Esch, M.H.M. Heemskerk, H. Ovaa and T.N.M. Schumacher. Conditional MHC class I ligands and MHC exchange technology
for the human MHC alleles HLA-A1, -A3, -A11 and -B7. Proc. Natl. Acad. Sci. USA Early edition. 10.1073/pnas.0709717105
3). G.M. Grotenbreg, N.R. Roan, E. Guillen, R. Meijers, J. Wang, G. W. Bell, M.N. Starnbach, and H.L. Ploegh. Discovery of CD8+ T cell epitopes in Chlamydia trachomatis infection through use of caged class I MHC tetramers Proc. Natl. Acad. Sci. USA Early edition. 10.1073/pnas.0711504105
RE: Is this technology truly preferable?
SorenBuus replied to Ton_Schumacher on 17 Mar 2008 at 21:06 GMT
Congratulations on the recent achievements. Please note that they were not in the public domain at the time of our paper being accepted.
Re. complexity: It is correct that the recombinant MHC molecules are purified in a multi-step procedure. However, there is nothing unusual about the complexity - or lack of complexity - involved in this purification strategy; the only unusual feature entails the omission of reducing agents during the purification.
Re. Yield (%): The Schumacher Nature Protocol (2006, 1:1120) paper states 5 to 12 % efficiency for the refolding process alone. Usually the overall efficiency of a multi-step process as the Nature Protocol one would be even lower. Our referenced yields (Ferre et al, Prot Sci 2002, 12:551) of 7 to 22% are for the overall process.
Re. Yield (mg): From a production point of view, a more interesting yield is that expressed as mg active material per production unit. Using a 2L lab fermentor (OD 25) we now routinely get about 150 mg fully active biotinylated MHC. By comparison, the 1L OD 0,8 culture used in the Nature Protocol paper yielded 0.2 ml of a 1.4 mg/ml biotinylated MHC - or 0,28 mg. Thus, our batch size is orders of magnitude larger, and our yield per bacterial cell is better.
Is our technology truly preferable? I would say YES. I see the future of MHC tetramer technology as being performed in two major steps:
A) Procurement: Large-scale production of biotinylated monomers will be done at a devoted facility. Our protocol generates large batches of fully active and fully biotinylated HLA heavy chains, which would be preferable whether one fold a conditional complex, as an intermediate towards the final complex such as Schumacher does, or whether one goes straight for the final complex, as we do. As alluded to above, our purification procedure is fairly standard. Furthermore, knowledge of an appropriate conditional peptide is absolutely essential for the Schumacher protocol and may limit the number of HLA molecules available using this technique. This concern does not apply to our approach.
B) End-use: The 1h exchange reaction of the Schumacher protocol would appear to give this technique an advantage in end-user friendliness. This loading rate, however, is obtained at very high peptide concentrations (50 micromolar, 1h, on ice). For comparison, we have loaded our HLA molecules under the same conditions. We obtained tetramers of staining quality fully comparable to those obtained in the paper using longer incubations periods.