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la d . SH3 NT 3064- 1018-

b)

Fig. 3.2 Construction of GST-Btk-SH3 fusion protein, a) The Btk-SH3

nucleotide sequence w as am plified by PGR using the primers SH3F and SH3R. The product of the NTF and NTR PGR reaction (Btk NT) is also show n. Reaction products were separated on a 1 % agarose gel containing ethidium bromide and visualised using UV light. The sizes of 1 kb ladder fragments are indicated in base pairs. PGR fragments w ere purified using N A 45 membrane, b) After transformation with pGEX-2T containing insert, ampicillin resistant £.

coli clones SH3-1, 2 and 3 were induced (+) with IPTG (0.1 mM) and lysed after 1 h. Lysates were resolved on a 15% SDS-PAGE gel and stained with CBB. The inducible protein is indicated by an arrow c) Clone SH3-1 w as induced to produce fusion protein. Cells were lysed, incubated w ith GS-4B beads which were then w ashed. A liquots representing various bead volum es w ere separated by SDS-PAGE. Known quantities of GST-Src-SH3 domain fusion protein w ere also electrophoresed to estim ate protein concentration. The size of molecular w eight markers is indicated in kDa.

CHAPTER 3_________________________________________________GST FUSION PROTEINS

0.1 m M IPTG for 1 h. A sample w as taken, boiled in SDS loading buffer and resolved by SDS-PAGE. The gel was stained w ith CBB to check for inducible protein of 34 kDa, the predicted size of the fusion protein. Fig. 3.2b shows that clones SH3-1 and 3 contained an inducible protein of this size, w hereas SH3-2 did not.

To ensure that no m utations had been generated by the PGR, plasm id w as purified from clones SH3-1 and 3 using the Magic M iniprep system

(Promega) and both strands of the insert sequenced using prim ers SH3F and SH3R. These sequences were identical to that published for the Btk SH3 dom ain and confirmed that the insert had been ligated in frame w ith the GST sequence (data not shown). Clone SH3-1 was chosen to express the GST-Btk- SH3 fusion protein.

3.3.3 Affinity purification of GST-BTK-SH3

Clone SH3-1 was grow n in 800 ml of L-broth and fusion protein purified using GS-4B beads as described in section 2.2.5. Fusion protein bound to GS- 4B beads was stored as a 10% m ixture (v /v ) in storage buffer. Aliquots of this m ixture w ere analysed using SDS-PAGE to determ ine concentration and purity.

Fig. 3.2c com pares purified GST-Btk-SFI3 w ith know n concentrations of GST- Src-SH3 fusion protein, allowing its concentration to be estim ated at 1

m g / ml GS-4B bead volume, giving a total yield of 7.5 mg. The CBB stain also detected a species running slightly faster than the m ain fusion protein band which could represent GST from degraded fusion protein or from cell

transform ed w ith w ild type pGEX-2T. A nother faint band m igrating at 70-80 kDa is observed which probably depicts a bacterial contam inant (section

3.6.2).

CHAPTER 3_________________________________________________ GST FUSION PROTEINS

3.4

PRODUCTION OF GST-BTK-SH2 MUTANTS

Analysis of cDNA from XLA patients JP and 276 had identified point m utations w ithin their Btk SH2 dom ains (Bradley et aL, 1994) (Fig. 2.1). Interestingly, com parison w ith know n SH2 dom ain structures (Waksman et al., 1993) suggested that the m utated residues lay in the phosphotyrosine binding pocket critical for the high affinity interactions observed betw een SH2 dom ains and their targets. The arginine substituted in JP (R307G) (residue pB5 - section 1.4.2.1) is invariant in all SH2 dom ains described so far and m utation of the equivalent residue to lysine in the Abl protein has been show n to completely prevent phosphotyrosine binding (Zhu et al., 1993). Patient 276 has an R288W m utation. This residue is not as highly conserved as R307, but is believed to contribute to the stability of SH 2/

phosphotyrosine binding (W aksman et al., 1993). The patient has a less severe phenotype than JP which may reflect a difference in the affinity of their SH2 dom ains for their targets.

These GST-SH2 m utants were constructed to act as negative controls for the previously generated wild type fusion protein. As they are predicted not to be able to bind phosphotyrosine residues, or to only bind them weakly, they w ere used to confirm that any observed wild type SH2 dom ain ligands were associating via the phosphotyrosine binding pocket.

The dom ains were amplified from patient cDNA using the PGR prim ers SH2F and SH2R (Table 2.2). The rest of the synthesis w as perform ed as described for the GST-Btk-SH3 fusion protein (section 3.3.1-3.3.3). Selected clones w ere sequenced to confirm the fidelity of the PGR and to ensure incorporation of the point m utations. Fig. 3.3a shows the m utated sequence for both JP and 276 fusion proteins. Fig. 3.3b shows purified m utant fusion protein of the same size as wild type GST-Btk-SH2, suggesting that these m utations do not affect the stability of the SH2 domain.

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