Ley de Contrato de Trabajo de 1944

system

3.1 Rationale for expressing DM in insect cells

A c tiv a tio n o f C D 4+ T cells by p e p tid e-lo a d ed M HC class II m olecules on the cell surface of antigen presenting cells requires a m u ltitu d e o f in tr a c e llu la r step s in th e g e n e ra tio n o f th e re c o g n itio n e le m e n ts. T h ese in c lu d e a h ig h ly s o p h is tic a te d in tracellu lar trafficking m achinery and at least tw o M HC class II en co d ed “u n c o n v e n tio n a l” m o lecu les th at e x e rt th e ir fu n c tio n s inside the cell. Those m olecules are DM and DO and their functions have only recently been elucidated.

The significance o f the presence of H LA-DM in the MHC class II a n tig e n p re s e n ta tio n p ath w ay b ecam e a p p a re n t w hen se v e ra l

d ifferen t m u tan t cell lines w ere an aly sed and fo u n d to sh are a s e v e re a n tig e n p r e s e n ta tio n d e fe c t. T h e ir p h e n o ty p e w as ch aracterized by an in ab ility to exchange C L IP fo r an an tig en ic p e p tid e in th e i n tr a c e llu la r p e p tid e - lo a d in g c o m p a rtm e n t. C onsequently m ost o f their surface class II m olecules w ere C L IP- loaded and could not induce a T cell response (C ressw ell, 1994). C o m p le m en tatio n stu d ies rev e ale d th a t the d e fe c t w as c au se d eith er by p o in t m u tatio n s in the DM A or D M B stru ctu ral genes (Fling et al., 1994; M orris et al., 1994) or their absence alto g eth er (D enzin and C ressw ell, 1995). T ransfection with the D M A and D M B

cD NA s led to a restoration of the w ildtype phenotype and the loss o f the CLIP fragm ent from the class II m olecules. This lead to the hypothesis that DM was involved in the rem oval o f CLIP from the M H C class II p e p tid e b in d in g g ro o v e in the p e p tid e lo a d in g com partm ents, thus m aking the antigen binding cleft available fo r o th er ligands. T here w ere several hypotheses about the m o lecu lar n atu re o f D M ’s action, for exam ple DM m ig h t have p e p tid ase activity and drive the equilibrium from class II a /P -C L IP to free a /p by degrading CLIP; it m ight act as a “CLIP sink” and bind CLIP its e lf or it m ight in teract with class II and som ehow induce the release of CLIP.

O ne objective o f this thesis was thus to analyze the fu n ctio n o f H L A -D M on a m o le c u la r lev el and to u n d e rs ta n d how th e displacem ent of CLIP is m ediated. W e seeked to set up an in vitro

system using purified DM and DR m olecules and a defined set o f p ep tid es to characterize their interaction in detail. Since H LA -D M is expressed in significantly lower am ounts than H LA -D R in B cells (D e n z in and C re ssw e ll, 1995; S c h a fe r et a l., 1996) and a m o n o c lo n a l a n tib o d y fo r larg e sc ale a ffin ity c h ro m a to g ra p h y p u rific a tio n w as in itia lly not a v ailab le we d e cid ed to e x p re ss

reco m b in an t DM using an in sect cell expression system th at had already been successfully used to express reco m b in an t D R l (Stern and W iley, 1992) and I-E and I-A m olecules (K ozono et al., 1994). In o rd er to fac ilitate the p u rific a tio n o f the re c o m b in a n t D M protein we decided on expressing a soluble version o f DM , lacking the transm em brane and the cytoplasm ic region o f both D M a and (3 th at w o u ld be secreted in to the tissu e c u ltu re su p e rn a ta n t by virtue o f the cleavable signal sequences at the N -term ini o f D M a and p. A hexahistidine-tag was genetically attached to the novel C- term inus of the recom binant DM p in order to m ake it possible to p u r i f y th e r e c o m b in a n t p r o t e i n v ia m e ta l c h e l a t i n g chromatography.

T he b acu lo v iru s ex p ressio n system has several ad v an tag es o v er o th er ex p ressio n system s. The in fected in se ct c ells ex p ress the heterologous genes in very high am ounts due to the exceptionally stro n g p o ly h ed rin an d /o r plO p ro m o ter(s) u sed in b a c u lo v iru s tran sfer vectors. The reco m b in an t p roteins are g en erally co rrectly co -tran slatio n ally m odified, and the low tem perature req u ired fo r the culture o f insect cells (27°C) prom otes the fo ld in g o f proteins that are otherwise difficult to express.

In o rd e r to g en erate re c o m b in a n t b a c u lo v iru se s the genes o f in te re st have to be cloned into a tran sfer v ecto r w hich co n tain s regions o f hom ology w ith the baculovirus genom e. C o -tran sfectio n o f in se c t cells w ith th ese co n stru cts and b a c u lo v iru s w ild ty p e genom ic DN A leads to hom ologous reco m b in atio n events du rin g w h ic h th e h e te ro lo g o u s g e n es are in c o r p o r a te d in to th e b a c u lo v iru s genom e. T he e ffic ie n c y o f th is p ro c e ss can be sig n ific a n tly enhanced by using b acu lo v iru s gen o m ic D N A th a t la c k s p a rt o f an e s s e n tia l o p en re a d in g fra m e so th a t

re c o m b in a tio n w ith the tran sfe r v e cto r c o n ta in in g the m is sin g fragment has to occur in order to generate viable viruses.

3.2 Cloning of truncated DMA and DMB cDNAs for

insect cell expression

For the ex p ressio n o f our DM co n stru cts we u sed a m o d ified version o f the dual-prom oter transfer vector pA cU W 51 (K ozono et al., 1994) in which the E co R I-B g /II restrictio n sites after the p lO p ro m o te r had been rep laced by the re s tric tio n sites 5 ' - X h o l - B s t B l l - M r o l - K p n l - V and the B a m Y i l re s tric tio n site afte r the p o ly h e d rin p ro m o te r by 5 ' - E c o B . l - S a l l - B g l l \ - S p h \ - B

(figure 3.1). The D M A and DMB c D N A s were altered by using the p o ly m e ra se ch ain re a c tio n (PC R ) so th at b o th cD N A s w e re tru n c a te d a fte r the c o n n e c tin g p e p tid e w h ic h a d jo in s th e m em b ran e -p ro x im a l dom ain to the tran sm e m b ra n e reg io n . T h e

D M A cD N A w as term in a ted a fte r bp 732 o f the p u b lis h e d se q u e n c e and D M B afte r b a se p a ir 822. S top c o d o n s w e re in tro d u ced into both sequences by PC R and a h e x a h istid in e -ta g preceded by a protein A cleavage site was attached to the novel 3 ’ end of the DMB sequence. For the cloning o f the truncated D M A

gene the X h o l and M r o l restriction sites dow nstream o f the plO p ro m o te r w ere used and for the D M B construct the E c o R I and

B g l l l site s d o w n stre a m o f the p o ly h e d rin p ro m o te r. T h e ap p ro p riate restrictio n sites were introduced into the sequence by PC R sim u ltan eo u sly w ith the other alteratio n s (fig u re 3.2). T he PC R products w ere digested with the above enzym es and clo n ed sequentially into pA cU W 51-’’Kozono” . The D M B fragm ent had to be cloned into the transfer vector first, otherw ise the D M A c o n stru c t w ould have been cleaved by Bgl W due to a B g l l l restrictio n site at

F i g u r e 3 . 1 B a c u l o v i r u s transfer v e c t o r - " K o z o n o " ( 5 . 8 kb