Ž .Journal of Immunological Methods 234 2000 83–88www.elsevier.nlrlocaterjim

Perfusion chromatography for very rapid purification of class Iand II MHC proteins

Pratap Malik, Jack L. Strominger )

Department of Molecular and Cellular Biology, HarÕard UniÕersity, 7 DiÕinity AÕenue, Cambridge, MA 02138, USA

Accepted 13 October 1999

Abstract

Ž .Major histocompatibility complex MHC proteins are surface glycoproteins that are strongly associated with either selfor foreign peptides. Their interaction with the T-cell receptor on the T-cells initiates an immune response and help indiscriminating between self and non-self, respectively. We describe here a novel means of rapidly purifying human MHCmolecules on either small scale or large scale from the cell lysate of lymphoblastoid B cell line and from insect cell culturesupernatants by using affinity perfusion chromatography. As representative cases HLA-B2705, a class I MHC molecule, andHLA-DR1, a class II MHC molecule were purified from EBV-transformed human lymphoblastoid B cells, LG2. SolubleHLA-DR1 was also purified from the cell culture supernatant of insect cells. The peptides eluted from the purifiedHLA-B2705 were pool sequenced and found to have the same motif as has previously been published. This new methodprovides a very rapid means of purifying MHC protein molecules, applicable to both large scale and small scale purification,which in turn greatly enhances the accuracy of further analysis of the associated peptides through mass spectrometry. q 2000Elsevier Science B.V. All rights reserved.

Keywords: MHC; Affinity-purification; Perfusion chromatography; Peptides

1. Introduction

Ž .Major histocompatibility complex MHC en-coded protein molecules are highly polymorphic sur-

Ž wŽ .AbbreÕiations: CHAPS 3- 3-Cholamidopropyl dimethylam-x .monio -1-propane-sulfonate ; DOC, deoxycholic acid; HLA, hu-

man leukocyte antigen; MHC, major histocompatibility antigen;Ž w x .MOPS 3- N-morpholoino propanesulfonic acid ; NMS, normal

mouse serum; PAGE, polyacrylamide gel electrophoresis; PBS,phosphate buffered saline; PBSA, PBS containing 0.05% sodiumazide; PEEK, polyetheretherketone; SDS, sodiumdodecylsulfate

) Corresponding author. Tel.: q1-617-495-2733; fax: q1-617-496-8351; e-mail: jlstrom@fas.harvard.edu

face glycoproteins that are involved in immunerecognition. The class I MHC molecule consists oftwo non-covalently associated chains — a 44,000-Datransmembrane a chain, and b2-microglobulin, a14,000-Da soluble protein. The class II MHCmolecule has two non-covalently associated trans-membrane chains, an a chain of 34,000 Da and a b

Ž .chain of 29,000 Da Jones, 1997 . Both class I andclass II MHC protein molecules are strongly associ-ated with a broad spectrum of peptides which arepresented to T cells via interaction with the T cell

Ž .receptor Engelhard, 1994; Madden, 1995 . T cellsdiscriminate between self and non-self depending

0022-1759r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.Ž .PII: S0022-1759 99 00201-X

( )P. Malik, J.L. StromingerrJournal of Immunological Methods 234 2000 83–8884

upon whether these peptides are of self or foreignorigin as a result of thymic selection. Three-dimen-sional structures of several class I and class II MHCmolecules have demonstrated the importance of thepeptide component of the complex in the recognitionprocess, which through the side-chains and confor-mational variability provides an antigenically uniquesurface of the MHCrpeptide complex to T cellreceptors. Several methods using immunoaffinitychromatography and mass spectrometry have beendeveloped to characterize naturally processed pep-tides presented by the class I and II MHC protein

Ž .molecules Joyce and Nathenson, 1994 . The purifi-cation of the MHCrpeptide complexes and the char-acterization and the sequence analysis of peptideshas provided a good understanding of the effects ofMHC polymorphism, the mechanism of antigen pro-cessing and presentation and the functional aspects

Ž .of MHC molecules Harris, 1994 .The most commonly used procedures for purifica-

tion of MHC molecules is based on immunoaffinityŽ .purification originally developed by Parham 1979

Ž .and Gorga et al. 1987 . These current protocols usehigh porosity cross-linked carbohydrate supports forexample, Sepharose. Since these supports are com-pressible, the purification using these supports isdone at slow flow rates over a period of 7–10 daysat 48C. Here a new methodology is described usingimmunoaffinity columns in perfusion chromatogra-

Ž .phy Afeyan et al., 1990 . Perfusion chromatographyinvolves the flow of liquid through a non-com-

Ž wpressible porous chromatographic particle POROS.Media, PerSeptive Biosystems with 6000–8000 A

pores which transect the particle. These throughporesallow very high flow rates and enable rapid loading,washing, cleaning and elution of the column. Here,an optimized protocol for the purification strategyfor class I and II MHC protein molecules whichincludes column generation, cell lysate preparationand isolation of the MHC protein molecule is de-scribed. Using this new methodology, one can gofrom frozen cells to pure MHC molecule in a matterof a few hours as opposed to 7–10 days. A decreasein purification time leads to higher purity and repro-ducibility. Also, one can purify protein ranging froma few micrograms to several milligrams using thismethodology.

2. Materials and methods

2.1. Monoclonal antibodies

The monoclonal antibodies used were ME1, ananti-HLA-B27 mouse IgG1 monoclonal antibodyŽ .Ellis et al., 1982 , and LB3.1, an anti-HLA-DR

Žmouse IgG2b monoclonal antibody Gorga et al.,.1986 . The ME1 hybridoma cells were grown in

Ž .Hybridoma Serum Free Medium Gibco BRL sup-Žplemented with 0–1% fetal bovine serum Gibco

. Ž .BRL , 2 mM glutamine Gibco BRL , 50 UrmlŽ .penicillin Gibco BRL and 50 mgrml streptomycin

Ž .Gibco BRL . The LB3.1 hybridoma cells weregrown in RPMI 1640 supplemented with 1% fetal

Ž .bovine Serum, 2 mM glutamine Gibco BRL , 50Ž .Urml penicillin Gibco BRL and 50 mgrml

Ž .streptomycin Gibco BRL . The monoclonal antibod-ies LB3.1 and ME1 were purified by running the cell

w Žculture supernatant on either POROS 20 A proteinw . wA coupled POROS 20 medium or POROS 20 G

Ž wprotein G coupled to POROS 20 medium used for.IgG1 antibodies columns, respectively using a Bio-

CADe Workstation for perfusion chromatographyŽ .PerSeptive Biosystems . Typically, 1–2 l of cellculture supernatant was filtered through 0.2 mmfilter and run on a POROSw 20 A or POROSw 20 Gcolumn. The column was washed with 5 column

Žvolumes of PBSA PBS containing 0.05% sodium.azide and eluted with about 0.5–1 column volume

of 2% acetic acid. The eluted antibody was immedi-ately neutralized with 1 M Tris base and the columnequilibrated with PBSA.

2.2. Immunoaffinity columns

Typically 10–20 mg of the purified monoclonalantibody in PBS was coupled to 1 ml of POROSw

Ž w20 AL medium POROS 20 medium activated with. Ž .the aldehyde group PerSeptive Biosystems . To

about 5–10 mgrml of antibody in PBS was addedŽ1r2 volume of High Salt Buffer Solution 1.5 M

sodium sulfate in 100 mM sodium phosphate pH.7.4 . This was made 5–10 mgrml in NaCNBH3

Ž .Sigma . To this was added the appropriate amountw Žof POROS 20 AL generally slightly more than the

.desired column volume and the solution was made

( )P. Malik, J.L. StromingerrJournal of Immunological Methods 234 2000 83–88 85

to 0.9–1.1 M in Na S0 by the addition of High Salt2 4

Buffer Solution. The final concentration of the anti-body was between 1 and 2 mgrml. The reaction wascarried out overnight by gentle shaking. The mediawas filtered in a 10–20 mm sintered glass funnel and

Žresuspended in 50–100 ml of Capping Buffer 5 grl.NaCNBH in 0.2 M Tris, pH 7.2 for about 1 h. The3

media was then washed with PBS and packed in aŽcolumn. Columns ranging from 4.4 ml 100=7.5

. Ž . Žmm to 13.25 ml 300=7.5 mm PEEK poly-. Ž .etheretherketone columns Alltech were packed un-

der the conditions specified by the manufacturer. Apre-clearing column using normal mouse serumŽ .NMS was also prepared and used to remove pro-teins that adhered non-specifically to IgG.

2.3. Preparation of MHC protein molecules

2.3.1. From cell lysateLG2 cells, Epstein Barr Virus-transformed human

B cells homozygous in HLA-B2705 and in HLA-DR1were grown in RPMI 1640 supplemented with 1–

Ž .10% fetal bovine serum HyClone , 2 mM glutamineŽ . Ž .Gibco BRL , 50 Urml penicillin Gibco BRL and

Ž .50 mgrml streptomycin Gibco BRL in roller bot-tles. About 1 l of culture gave approximately 1 g ofwet weight of cells. Cells were pelleted, washed withPBS and kept frozen at y708C until they were used.Cells were thawed and resuspended in 5–10 vol-

Žumerwet wt of Lysis Solution 1% Nonidet P-40,150 mM NaCl, 20 mM Tris–HCl pH 8.0, 0.1 mM

.PMSF with vigorous vortexing. The lysate wasŽcleared by centrifugation at 15,000=g 9009 rpm in

.a Beckman JLA 10.500 rotor for 0.5 h and then byŽultracentrifugation at 150,000=g 35,900 rpm in a

.Beckman 45 Ti rotor for 1.5 h. The lysate was thenŽ .filtered through 0.8 mm filter Nalgene and then

Ž .through 0.2 mm filtration unit Corning . The clearedlysate was run through a series of columns in theorder POROSw 20 AL-NMS, POROSw 20 A, andPOROSw 20 AL-LB3.1 andror POROSw 20 AL-ME1 at a flow rate of 5 mlrmin. Each column waswashed separately with 10 column volumes each of

ŽWash Solution I 0.1% Nonidet P-40, 10 mM Tris–. ŽHCl pH 8.0 , Wash Solution II 0.1% DOC, 140 mM

.NaCl, 20 mM MOPS pH 8.0 and Wash Solution IIIŽ .0.1% DOC, 10 mM Tris–HCl pH 8.0 . ThePOROSw 20 AL-NMS was washed with 5 volumes

Žeach of PBS, Elution Solution 0.1% DOC 50 mM.glycine pH 11.0 , PBS, 2% acetic acid and PBS at a

flow rate of 10 mlrmin. The POROSw 20 A columnwas washed with 5 volumes of PBS and then with2% acetic acid solution and with a further 5 columnvolumes of PBS. The MHC protein was eluted fromPOROSw 20 AL-ME1 or the POROSw 20 AL-LB3.1

Žcolumns by passing the Elution Solution 0.1% DOC.50 mM glycine pH 11.0 at a flow rate of 2–5

mlrmin. The eluted protein was immediately neu-tralized with 2 M glycine pH 2.0 and dialyzedovernight against 0.1% DOC, 10 mM Tris–HCl pH8.0. After eluting the proteins several column vol-umes of PBSA were passed through the column at10 mlrmin.

2.3.2. Recombinant proteins from cell supernatantSoluble HLA-DR1 molecules were expressed in

ŽDrosophila S2 cells as described Stern and Wiley,.1992; Kalandadze et al., 1996; Dessen et al., 1997 .

Cells were grown in roller bottles in ExCell 401Ž .medium JRH Biosciences supplemented with 0–5%

Ž .fetal bovine serum Sigma at 268C. Cells wereharvested 4–5 days after induction by 1 mM CuSO .4

The supernatant was collected by centrifugation, fil-Ž .tered through 0.2 mm filtration unit Corning . The

filtered supernatant was passed through the series ofcolumns consisting of POROSw 20 AL-NMS,POROSw 20 A, and POROSw 20 AL-LB3.1. Theprotein was eluted by passing 50 mM glycine pH11.5 solution at 2–5 mlrmin through POROSw 20AL-LB3.1 column. The column was washed withseveral volumes of PBS and the eluted protein wasimmediately neutralized with 2 M Tris–HCl pH 6.5.The POROSw 20 AL-NMS and POROSw 20 Awere treated as above. Typically 1 l of cell super-natant was used with columns of 13.25 ml.

2.4. Peptide isolation

The lysate was prepared and run on the columnsas above except for the elution step. The POROSw

20 AL-ME1 column was washed with 5 columnvolumes each of 1% CHAPS, 150 mM NaCl, 20 mMTris–HCl pH 8.0; 150 mM NaCl, 20 mM Tris–HClpH 8.0, 1 M NaCl, 20 mM Tris–HCl pH 8.0; 20 mMTris–HCl pH 8.0. The MHC proteinrpeptides wereeluted with 2% acetic acid. The eluant was made

( )P. Malik, J.L. StromingerrJournal of Immunological Methods 234 2000 83–8886

10% in acetic acid and concentrated by evaporationin a speedvac. 400 ml of the concentrated mixture

Ž .was loaded on 1.2 ml Bio-Gel P2 gel Bio-Rad ,pre-equilibrated with 10% acetic acid in a Bio-Spin

Ž .Column Bio-Rad . The column was washed with500 ml of 10% acetic acid to collect the larger

Žproteins the a chain of the HLA protein and b2-mi-.croglobulin . The peptides were eluted by a further

wash with 2 ml of 10% acetic acid. The peptidecontaining fractions were pooled, concentrated byevaporation in a speedvac and pool sequenced. About5000 pmol of peptides were obtained from the HLA-B2705 molecules from 10 g cells as determined byamino acid analysis of the eluted peptide pool.

3. Results and discussion

Perfusion affinity-chromatography using the Bio-CADe Workstation has been applied for very rapidpurification of MHC class I, HLA-B2705 and classII, HLA-DR1 protein molecules, from Epstein BarrVirus-transformed human lymphoblastoid B cells.With this new methodology the time from frozencells to pure MHC molecules is reduced to about 3 hfor small scale to about 6 h for large scale in contrastto the traditional chromatographic techniques which

Fig. 1. SDS–PAGE analysis of purified HLA-B2705 and HLA-Ž .DR1 protein molecules from 100 g cells large scale and 10 g

Ž .cells small scale and from cell supernatant of soluble HLA-DR1producing cells. The gel was stained with Coomassie blue.

Table 1Yields of MHC proteins

Ž . Ž .Small scale 10 g cells Large scale 100 g cells

HLA-B2705 0.5–1 mg 7–10 mgHLA-DR1 5–10 mg 50–75 mg

take about 7–10 days. The yield of HLA-B2705obtained was about 0.5–1 mg from 10 g of cells andthe purity is greater than 80% as seen by SDS–PAGEŽ . Ž .Fig. 1 Table 1 . Peptides isolated from thePOROSw 20 AL-ME1 column purified HLA-B2705were pool sequenced and shown to conform to theexpected motif of arginine at position 2, and tyro-

Žsine, phenylalanine or leucine at position 9 Jardetzkyet al., 1991; Rotzschke et al., 1994; Urban et al.,

. Ž .1994 Table 2 . The yield of HLA-DR1 was about 5mg from 10 g of cells which is comparable to the

Ž . Žpreviously reported yields Gorga et al., 1987 Fig.. Ž .1 Table 1 . The technique was also successfully

applied towards the purification of soluble emptyŽ .HLA-DR1 from cell culture supernatant Fig. 1 . In

addition to the 10 g scale, this technique worked

Table 2Pooled sequencing of HLA-B2705 eluted peptidesThe yield in pmol of amino acid residues in each sequencing cycleof Edman degradation is shown. The yields for the expected motifviz. arginine at position 2, and tyrosine, phenylalanine or leucineat position 9 are shown in bold

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

N 3 2 13 12 7 9 9 22 4 2 2 1 1 1 1S 7 5 5 8 6 7 5 6 3 2 5 3 2 2 1Q 1 21 10 29 13 14 8 18 9 4 2 2 2 2 1T 5 3 7 22 20 12 16 12 5 5 3 2 2 2 2G 32 8 8 27 33 14 11 14 11 6 4 3 3 3 3E 3 7 6 26 20 17 14 21 8 5 3 3 3 3 3H 3 2 5 4 4 4 4 5 4 3 2 1 4 4 3A 34 12 12 16 14 26 15 17 8 5 5 4 3 3 6R 94 285 83 33 21 20 15 21 18 15 10 14 9 8 5Y 7 2 21 7 10 7 12 7 11 19 8 4 3 2 2P 6 5 17 13 21 19 14 8 5 3 3 2 2 6 8M 3 4 8 5 6 2 3 2 7 3 2 1 1 1 0V 7 7 15 6 14 16 17 11 15 7 4 4 3 2 2F 11 4 43 9 12 8 16 8 17 8 4 2 2 2 1I 15 3 12 3 6 6 10 4 4 2 1 1 1 1 0K 14 2 9 12 10 14 6 7 13 5 3 1 1 1 1L 5 6 32 14 16 14 24 13 34 10 5 3 2 2 2W 1 0 8 3 2 1 3 1 1 1 0 0 0 0 0D 5 4 7 27 16 11 0 8 5 4 3 3 3 2 2

( )P. Malik, J.L. StromingerrJournal of Immunological Methods 234 2000 83–88 87

equally well with a scale up to 100 g of cell cultureand a scale down to 1 g of cell culture. Thus thismethod is potentially applicable to the isolation ofMHC protein and identification of peptides fromsmall amounts of material from autoimmune sites forexample from human synovia removed at synovec-tomy.

In the above purification protocol antibody wascoupled to POROSw 20 AL medium. This waspreferred over coupling the antibodies via the Fcreceptor to protein A or protein G conjugated mediaŽ w w .POROS 20 A or POROS 20 G since the bind-ing capacity of the POROSw 20 A media variedconsiderably with the antibody being bound. Thealdehyde chemistry was therefore used for couplingthe antibody to the POROSw 20 AL medium. Thisprocedure also has the advantage of eliminating thepossibility of any unwanted binding of antibodiespresent in the cell lysate to the unsaturated protein Asites on the protein-A medium. The cells were lysedin Nonidet P-40. However, as this detergent absorbsstrongly at 280 nm, it was exchanged with deoxy-cholic acid. For peptide elution at low pH the DOCwas exchanged with CHAPS as DOC has low solu-bility below neutral pH.

MHC-bound peptides are critical in the function-ing of the immune system. In recent years severaltechniques have been developed for sequence analy-sis of peptides at picomolar to subpicomolar levelsfrom mixtures bound to MHC molecules includingthe use of ion trap mass spectrometry and electro-

Žspary ionization tandem mass spectrometry ESI-. Ž .MSrMS Hunt et al., 1992a,b and automated data

acquisition and computer assisted interpretationŽ .Yates et al., 1995; Dongre et al., 1997 . However,the very first step in this analysis, viz isolation of theMHC molecules from cells and tissues has not im-proved since its introduction 10–20 years ago. De-velopment and modernization of the methodologiesof purification and isolation of MHC proteinmolecules and the associated peptides was needed tokeep pace with the state-of-the-art technologies forprotein purification and peptide analyses. Themethodology described above serves as a model forthe rapid purification of all other MHC class I andclass II molecules. The speed of purification reducesthe handling time of the protein and ensures animproved quality and a more authentic analysis of

the MHC–peptide complex. Such a technologicaladvance is fundamental to a sophisticated study ofthe immune response to foreign antigens, self-toler-ance and autoimmunity and to the development ofpeptide vaccines based on the use of MHC-restrictedepitopes for anti-tumor and anti-viral immunother-apy.

Acknowledgements

We would like to thank Dr. W.S. Lane for poolsequencing of peptides, Mrs. P. Klimonovitsky fortechnical assistance and Mrs. M. Mandelboim forgrowing HLA DR1 producing insect cell culture.This work was supported by the NIH contract NI-AID-NO1-A1-45198.

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