Diseño de los microaspersores

100|ig samples of F9 nuclear extracts were run on a single gel, and following transfer the filter was cut into four panels. Tw o w ere hybridized overnight with wild-type and mutant G S T C R E B (unlabelled) p repared from the p G E X -2TK vector, using the Far W estern

procedure, then subjected to Western analysis as described in Chapter

2 but using only 0.0 5 % Tw een-20 in PBS throughout (W W w t and

W W m t). Another panel was probed with the wild-type 32P -G S T C R E B shown in fig4.2c and the final panel underwent W estern analysis in 0.05% Tween-20/PBS.

FW, Far Western. W W , Wild Western. W, Western, wt, wild-type

G S T C R E B . mt, G S T C R E B L 1 2 3 4 V . U, undifferentiated and D, differentiated F9 nuclear extracts. Cr, baculovirally expressed CREB.

C R EB control detected by Wild Western or the 45 kD factor detected in F9 extracts by Western analysis. The single 97 kD band observed in figure 4.1c appears in figure 4.2d to be replaced by two bands, one smaller and the other significantly larger than 97 kD, although precise mobilities are difficult to estimate in this size range. Overall, it seems that the Wild Western blot is potentially more sensitive than the Far Western, since background problems are reduced. In this particular case, however, the interpretation of results must be hindered by the limitations of our antibody, which appears to cross-react with some of the 'CREB-interacting factors' seen on the blot. Nevertheless, it is clear from figure 4.2d that many of the interactions observed are dependent on the intact leucine zipper; only those in the range 90-200+ kD may be leucine zipper-independent, since they are better detected in the Wild Western with mutant C R E B than in the control Western. The cross-reaction of our antibody with the 30-35 kD doublet previously observed reinforces our speculation that this is CREM , since the C-termini of CREB and CREM (DBD I) are highly similar (10 consecutive residues in the 12 residue CREB peptide recognized by the antibody are identical).

Since the low yields of G STCR EB (either wild-type, or full-length mutant forms) continued to be a problem, an alternative method for expressing G S T fusion proteins in bacteria was introduced, as described in the following section.

4.1.iii GSTCREB labelled by phosphorylation

The RNA polymerase from bacteriophage T7 is very active, showing an elongation rate five fold faster than that of the E.coli polymerase. It can also squelch the activity of the host polymerase by successful competition for limiting factors and thereby has the ability to direct virtually all transcriptional activity in host cells to T7-specific promoters (pET vector series, Novagen). Since the yield of GSTCREB - as of other DNA-binding factors - from E.coli\s

diminished by its adverse effect on the health, or growth, of IPTG-induced cultures, one strategy for improving the yield of such proteins is to shorten the induction protocol and reduce the exposure of expressing cells to the foreign protein. T7-based expression systems, by directing rapid accumulation of products, allow induction times to be significantly shortened. An additional benefit of this system derives from the use of T7 lysozyme, which is introduced into some host strains (protease-deficient BL21) on a plasmid conferring chloramphenicol resistance. The lysozyme not only facilitates the preparation of bacterial lysates but also acts as an inhibitor of T 7 polymerase. This increases the tolerance shown by the host strain for toxic target plasmids by

providing repression of T7 polymerase prior to IPTG induction (Studier et al., 1990); the inhibition effect is titrated out on IPTG-induction of the polymerase.

Sequences from pG E X -2T K C R E B (197-341) coding for the G S T- fusion protein were cloned as described in the legend to figure 4.3a into a pET3-derived vector (pRK172 (McLeod et al., 1987)) (pRKGEXCREB, fig4.3a) and expressed in BL27.plysS. Figure 4.3b shows G S T C R E B (wild-type and mutant) and the G ST fusion of homeobox protein H IC(91-243) (Chapter 6) both

in whole bacterial lysates and following purification by G S H -affin ity chromatography. The yields are approximately lOOpg per 200ml of culture, 10- fold greater than those achieved using the pG EX vectors. Figure 4.3b illustrates again that a much larger yield is obtained from the protein which does not bind DNA (zipper mutant of CREB) as opposed to the two which do; also that the mutant protein is more readily eluted from glutathione-agarose.

32p-iabelled C-terminal fragments of C R EB wild-type, L2V and L I2 3 4 V were generated from the pRK vector, by thrombin treatment and PKA- phosphorylation of G ST fusions as described in Chapter 2. These C REB (197- 341) probes were hybridized to blotted extracts (fig4.3c). Figure 4.3c confirms observations made previously: That CREB interactions appear to occur with nuclear factors only, that there is one or more strongly interacting factor(s) of approximately 97 kD in size which appears to be less abundant in D F9 cells than in UF9 cells, and that an interacting factor at 70 kD is similarly down- regulated upon differentiation (possibly CREB2 or H SP70). A factor of the sam e mobility as C R EB shows some increase in abundance in the differentiated extracts: These results are the first of this chapter to show that it is possible to detect endogenous CREB - and probably ATF-1 also - in extracts, by Far Western. Figure 4.3c (and later, figures 4.4c&d) also departs from the pattern previously observed in that the number and the relative strength of interactions seen with large factors (over 97 kD) is greatly increased, possibly due to the use of phosphorylated probes (see section 4.2.Ü). Figure 4.3c seeks to investigate the effect of mutations in the leucine zipper on the pattern of interactions seen with F9 extracts. Contrary to the published data available at that time, CREB bearing the leucine 2 to valine substitution (L2V) was shown to maintain all of the interactions undertaken by the wild-type protein, since this mutant was able to compete to some extent with the wild type probe for all of the interactions seen (centre panel). However, when labelled and used as a probe, the L2V protein appeared to interact less well (although all of the same interactions were detectable) than might be expected from the competition result. W e concluded that although care was taken to ensure that equal amounts of full-length probe was hybridized to each blot, the greater instability of the mutant probes as compared to the wild-type (fig4.2c) was giving rise to

Figure 4.3a Phosphorylatable GSTCREB in a TT-directeci