MODELO RACIONAL

The full open reading frame was assembled by ligating together the 5’ RACE product, the A5 Jurkat library clone and the 3’-end from the z20468 probe. PCR was then used to incorporate a 5' Bam HI restriction site into the full length PtdlnsPK I la cDNA. The open reading frame was fully sequenced and expressed as an N-terminal GST fusion protein in pGEX-2T. Affinity-purified recombinant proteins were analysed by SDS-PAGE. The GST fusion protein had an apparent molecular mass of 79 kDa, comprising the 46 kDa kinase and the 26 kDa GST domain (Figure 3.4). As shown in Figure 3.4, the majority of GSTxPtdlnsPK I la was insoluble. As well as the major 79 kDa protein, preparations were also found to contain a number of lower molecular mass proteins likely to be fragments of the fusion protein.

3.2.4 Activity

Purified GST fusion proteins were eluted with free glutathione and assayed for kinase activity against PtdlnsP. The products of this reaction comigrated with [^H]-PtdIns(4,5)P2

91 A B Immune Pre-immune G L G L GST::PtdlnsPK Iia 53 kDa

Figure 3.3 Antiserum raised to synthetic peptide detects a 53 kDa protein in cell lysates

A . Serum from a rabbit im m u n ised w ith the sy n th etic peptide L 4, or p re-im m u n e serum from the sa m e rabbit w as used to probe identical w estern blots c o n ta in in g recom b in an t G S T ::P td In sP K I l a (G ) or a p p ro x im a tely 5 0 |ig o f a total Jurkat c e ll ly sa te (L ). B Im m u n e serum w a s su b seq u e n tly a ffin ity p u rified on the p e p tid e im m u n o g e n im m o b ilis e d on A ff ig e l resin ( B io R ad , s e e S e c tio n 2 .5 .2 .d ) and u sed to probe Jurkat c ell ly sa tes.

Mw (kDa) 97 - 67 - 45 - 30 - # G S T : :PtdlnsPK I l a GST

Figure 3.4 Recombinant expression of PtdlnsPK I la in E. coli.

The full length PtdlnsPK Ila cDNA was cloned into pGEX-2T (Pharmacia) and expressed as an N-terminal fusion protein in E.coli XL 1-Blue. 2 ml logarithmic-phase cultures containing pGEX-2T: : PtdlnsPKIIa or parental vector (control) were induced with 0.1 mM IPTG for 3 h at BO^C. Cells were collected by centrifugation and sonicated in lysis buffer (100 mM Tris.HCl, pH 7.4, 100 mM KCl, 1% Triton X-100, 10 mM P- mercaptoethanol, 1 mM EDTA, 1 mM PMSF, 1 mM benzamidine, 1 pg/ml aprotinin, 1 pg/ml leupetin and 1 pg/ml poly(L)lysine). Lysates were cleared at 18,000 xg and the supernatant applied to GSH-Sepharose resin (Pharmacia) previously equilibrated in lysis buffer, and incubated for 20 min at room temperature. After washing three times in lysis buffer the affinity-purified proteins were analysed by SDS-PAGE. Lane 1; pGEX-2T soluble fraction, lane 2; pGEX-2T insoluble fraction, lane 3; pGEX-2T: : Ptdlnsf K lla insoluble protein, lane 4; pGEX-2T: : PtdlnsPKIla affinity-purified protein.

93 Ptdlns(4, 5)P2 lysoPtdlns(4,5)P2 Origin [3H]PtdIns(4,5)P2 B signal (mV) 50.00 40.00 Ptdlns{4, 5)P; Standards 30.00 PtdlnsPK Ila PtdIns(3,4)P; 2 0 . 0 0 PtdIns(4)P 1 0 . 0 0 0 . 00 0 20 40 60 80 10 0 elution time(minutes)

Figure 3.5 The isolated cDNA encodes a PtdlnsP kinase

A p p ro x im a te ly 100 ng o f G S T fu sion protein w a s e lu ted from O S H -S e p h a ro se w itli 10 m M glu tatliion e and a ssa y e d for P td ln sP K a ctivity. R eaction s w ere p erform ed in 5 0 m M T ris.H C l, pH 7.4, 100 m M K Cl, 10 m M M g C h , 5 0 m M P td ln sP (S ig m a ), 2 0 p M A T P and 5 p C i o f y[32p ]A T P . A s sa y s w ere started by (lie a d d ition o f A T P , in cu b a ted for 15 m in at 3 7 °C and term inated by a d d in g H C l to 5 0 0 m M . T he organ ic ph ase w a s extracted and tlie lip id products separated by T L C as d escr ib ed in C hapter 2. T h e P td ln sP i sp o ts w ere scraped from die T L C plate and a n a ly sed by d é a cy la tio n and H PLC as describ ed p rev io u sly (Serunian et ai, 1991).

A . A u torad iogram o f the reaction products ch rom atograp h ed a lo n g sid e a [^ H ]-P td ln s(4 ,5 )P 2 standard (1, G S T ;:P td ln s P K lla reaction; 2, G S T control). B. H PLC h ead grou p a n a ly sis o f die d ea c y la te d reaction products.

when separated by TLC. Déacylation and headgroup analysis by HPLC revealed a single product which coeluted with a GroPtdIns(4,5)P2 standard (Figure 3.5), thereby confirming that the ORF encoded a PtdlnsPK. GST::PtdInsPK I la activity was not stimulated by PtdOH under conditions that activated recombinant human PtdlnsPK Ia/STM7 (data not shown).

3.2.5 Recombinant expression in S. frugiperda cells

Expression of PtdlnsPK Ila in bacteria yielded a population of partially truncated proteins which are most likely the result of proteolysis and/or premature translational termination due to overexpression of rare codons in E. coli (Figure 3.6, note that this phenomenom is clearer in other experiments, e.g. Figure 4.6). In order to overcome this problem and to improve the yield, solubility, and specific activity of recombinant protein for later structural studies, the PtdlnsPK I la ORF was cloned into the baculovirus transfer vector pAcG-2T. Sf9 cells were co-transfected with the transfer vector harboring the PtdlnsPK I la ORF and Baculovirus DNA (BaculoGold, Invitrogen) prior to the generation of a high titre stock (Section 2.3.2).

A time course study of the expression of GSTxPtdlnsPK I la in baculovirus-infected Sf9 cells was used to determine that the optimum yield of soluble protein occurred at 72 h post infection (data not shown). Purification was also analysed by western immunoblotting (Figure 3.6) using affinity-purified aL4 antisera in order to evaluate the efficiency of the purification method, the purity of the protein and the extent of proteolytic degradation. Importantly, baculovirus-expressed protein typically exhibited a higher specific activity over its bacterial counterpart (data not shown), a phenomenom that may result from the presence of inhibitory fragments or a lack of a post-translational modification in the E. coli preparations. As expected, yields of protein from insect cells greatly exceeded those from E.coli preparations (typically 20 mg/1 and 200 fig/1, respectively) and very little degradation was apparent in the Sf9 preparations (Figure 3.6).