This ch ap ter shows that it is po ssib le to p ro d u ce soluble D M p r o te in in in s e c t c e lls th at se em s to sh a re m o st o f th e characteristics o f the m am m alian protein. The reco m b in an t alp h a and beta chains form a heterodimer o f the expected size w hich is secreted into the tissue culture supernatant. The dim er is stable w ithout the addition o f peptide, consistent with the concept that in spite o f the stru ctu ral sim ilarity to c o n v e n tio n a l class II m olecules, the function o f DM m ight not be to present peptides but rather affect the intracellular loading of classical MHC class II molecules, an idea that will be explored in the next chapter.
The reco m b in an t DM m olecule is fully glycosylated, does re a c t w ith le n til-le c tin sepharose and seem s m o stly E n d o H -re sista n t, unlike its m am m alian counterpart w hich rem ains partly E ndoH - sensitive, like DR, even in the m ature state. O ur results are in c o n t r a s t to r e p o r ts by S te rn a n d W ile y w h o d e s c r i b e d b a c u lo v ir u s - p r o d u c e d D R l m o le c u le s th a t r e m a in e d E n d o H - sen sitiv e (Stern and W iley, 1992). The expression levels o f the heterologous proteins (1-2 mg/litre) in this report were, how ever, m uch hig h er than ours (300 p g /litre) so that in their case the g l y c o s y l a t i o n m a c h in e r y m ig h t h a v e b e e n o v e r l o a d e d . In conclusion the glycosylation pattern o f the baculovirus-DM protein does not seem to be grossly altered.
T h e y ield s o f re c o m b in a n t DM th at we o b ta in e d u sin g the b a cu lo v iru s expression system were rath e r low (0.3 mg /litre), according to the literature expression levels of up to 100 m g o f r e c o m b in a n t p ro tein per litre of in se c t cells can be a ch iev e d (O 'R eilly et al., 1994). The reason for the d isc rep a n cy in the
o b se rv ed and the an ticip ated p ro tein y ield is n o t q u ite clear. Conceivably, efficient folding anc) p a ir i n g o f DM requires additional chaperones which are not present in insect cells. A lternatively, the le v e l o f t r a n s c r i p ti o n m ig h t h a v e b e e n c o m p r o m i s e d by unfavourable sequences in the coding or the 5 ’- or 3 ’-untranslated s e q u e n c e s . C e rta in s e q u e n c e s (T A A G and C T T A ) in th e hetero lo g o u s gene in the sense or antisense d irectio n have been reported to have an influence on the levels o f m R N A (Ooi and Miller, 1991). We have not checked the DM sequences rigorously for such sequences, these effects can therefore not be excluded. H ow ever, even though our expression levels o f the soluble DM w ere u n e x p e c te d ly low, the p ro te in seem ed to h a v e a c tiv ity sim ilar to the wildtype protein (see next chapter). Besides we did n o t o b se rv e m uch ag g re g atio n o f the r e c o m b in a n t p ro te in as reported by other researchers expressing DM in a different insect cell expression system with m uch higher protein yields (Sloan et al., 1995), although we found that the a d d itio n o f d e te rg e n t f a c ilita te d the p u rific a tio n o f r e c o m b in a n t D M and p re v e n te d precipitation of concentrated, purified material.
In conclusion the purified protein appeared to form a native-like D M a / p heterodim er and seemed to be an adequate substrate for further functional studies.