Human recombinant full-length wild-type IRAK-1 in the pcDNA3 vector (Invitrogen) was a kind gift fi’om Fillipo Volpe (GlaxoWellcome). The cDNA was originally obtained by RT-PCR fi’om messenger RNA isolated fi’om a HEK293 cell line (Volpe et al., 1997). All the IRAK I variants goierated fi’om this cDNA for this set of experiments were cloned into EcoRI/Xbal digested pBKRSV (Stratagene) (Figure 3-7). The pBKRSV vector was dephosphwylated using CLAP before cloning to prevent religatim of the vector (Section 2.2.7). The pBKRSV vector was chosen for these studies as it contains an RSV promote to drive eukaryotic expression. The activity of promoters varies considerably and it has long been established that the human cytomegalovirus (CMV) promoter is much stronger than the Rous sarcoma virus (RSV) promoter (Boshart et a l, 1985). Although overexpression studies are useful in the functional analysis of components of signalling pathways, they do suffer from the general problem that they may interact non- specifically producing efifects that do not mimic the physiological functions of the molecules. To try to reduce non-specific effects of the lRAK-1 constructs we wanted low levels of expression, just above physiological conditions in the reporter gene assays. This was achievable using tiie RSV promoter. Additionally, we wanted to determine the effects of the pBKRSV lRAK-1 constructs on IL-ip and TNFa- driven N FkB activaticm using an N FkB reporter gene assay. As the CMV promoter contains kB responsive elements, it was decided to avoid using this vector in case it interfered with results of the reporter gene assay. The kB sites within the CMV promote could compete with the reporter gene for endogenous N FkB
or altonatively, N FkB activation itself may affect expression levels of constructs in cytokine activated cells, but not in the controls, so the results would not be directly comparable.
3.3.2 Generation o f the [IRAKl-K(212-532)], [IRAK1-C(533-712)] and
|TRAK1-KC(212-712)] Constructs
A cDNA encoding the C-terminal 180 amino acid residues of human IRAK I cDNA was amplified by PCR using Taq polymerase and fiill-length wild-type IRAK in pcDNA3 as a template. The primers used (Cl and C2, see Table 3-2), were designed to incorporate EcoRl mà.Xbal restriction sites into the PCR product at the amino and carboxy-terminal ends respectively.
This enabled the PCR product to be ligated directionally into the mammalian expression vector pBKRSV in compatible restriction sites located within the multiple cloning site (MGS). EcoRl and Xbal restriction analysis was used to check that the cloning of the ~540bp IRAK C-terminal fragment had been successful. The [IRAKl-K(212-532)] and [IRAK 1-KC(212-712)] variants were also constructed by inserting PCR- generated cDNA fragments into pBKRSV. The primers used for these reactions were K1/K2 and KC1/KC2 respectively (Table 3-2). In all cases, sequencing (see section 2.2.9) using the T3 and internal IRAK I primers listed in table 2-1 was used to confirm the success of the cloning.
SV40 polyl Neo / Kan lacZ gene
pBKRSV 4 4 5 2 bps lac promoter Xbal, 1056 EcoRI,1083 RSV promoter TKpoly(A) ColEI o
Figure 3-7: T h e pB K R S V V ector (S tratagen e). The vector contains an RSV-(long-terminal repeat) LTR promoter
(1829-1305bp ) to drive eukaryotic expression and an SV 40 polyadenylation fragment (750-469bp ) that provides the
signal required for termination o f transcription and polyadenylation. Prokaryotic expression is driven by the lac promoter and is repressed in the presence o f the L ad protein. The multiple cloning site (M CS) contains 17 unique sites
and allow s both eukaryotic and prokaryotic expression. It is placed downstream o f the lac promoter (1300-1220bp ) in the amino terminus o f the a-com p lem enting portion o f the P-galactosidase gene (1183-81 Obp), allow ing blue/white
colour screening o f clones with inserts follow ing prokaiyotic expression induced with IPTG. For eukaryotic
expression, the insert must contain an ATG and Kozak sequence. Eukaryotic and prokaryotic selection is made
possible by the neomycin/kanamycin gene (3% 9-2967) which is driven by the SV 40 early promoter (4327-3970bp ) or
the P-lactamase promoter respectively. I he vector was digested with EcoRI and Xbal and dephosphorylated using ca lf intestinal alkaline phosphatase (CIAP) to allow directional cloning o f the IRAK-1 variants.
Cl CGG AATTC ACCATGG AGGCCGCC AGCTGC C2 CAGAGCTGATGTGTTCACCTGTCTAGATCC Ki CGGAATTCACCATGTTCTCGGAGGAGCTCAAGATCGG K2 GCTCTAGATCACAAATGCCCGGGCAC NI GGGCACCCACAACTGATCGGAGGTACCCAAGATCGGGGAGG N2 CCTCCCCGATCTTGGGTACCTCCGATCAGTTGTGGGTGCCC N Cla CGGGGCACCGATATCTTCTCGGAGGAG NClb CTCCTCCG AG AAG AT ATCGGTGCCCCG NC2a GTGCCCGGGGATATCGAGGCCGCCAGC NC2b GCTGGCGGCCTCCAAATGCCCGGGCAC NKl GCCCGGGCATTTGTGAGGTACCAGCTGCATCCCCCCTTCCCCG NK2 CGGGGAAGGGGGGATGCAGCTGGTACCTCACAAATGCCCGGG NFla GGATCCAAAGAATTCACCATGGACTACGCCGGGGGGCCGGGCCCG NFlb CGGGCCCGGCCCCCCGGCGT AGTCCATGGTGAATTCTTTGG ATCC
NF2a AAAGAA I TCACCATGGACI ACAAGGACGCCGGGGGGCCGGGCCCG
NF2b CGGGCCCGGCCCCCCGGCGTCCTTGTAGTCCATGGTGAATTCT'IT
NF3a GAATTCACCATGGACTACAAGGACG.4CGATGCCGGGGGGCCGGGC
NF3b GCCCGGCCCCCCGGCATCGTCGTCCTTGTAGTCCATGGTGAATTC
NF4a ACCATGGACI.\CAAGGACG,\CGATGACAAGGCCGGGGGGCCGGGCCCG
NF4b CGGGCCCGGCCCCCCGGCCTTGTCATCGTCGTCCTTGTAGTCCATGGT
KFla G A ATCC AAAG A ATTC ACC ATGG .‘\C T ACTTCTCGG AGG AGCTC A AG ATCGGGG AG
KFlb CTCCCCGATCTTGAGCTCCTCCGAGAAGTAGTCCATGGTGAATTCTTTGGATCC
KF2a AAAGAATTCACCATGGACTACAAGGACTTCTCGGAGGAGCTCAAGATCGGGGAG
KF2b CTCCCCGATCTTGAGCTCCTCCGAGAAGTCCTTGTAGTCCATGGTGAATTCTTT
KF3a GAATTCACCATGGACTACAAGGACG.ACGATTTCTCGGAGGAGCTCAAGATCGGGGAG
KF3b CTCCCCGATCTTGAGCTCCTCCGAGAAATCGTCGTCCTTGTAGTCCATGGTGAATTC
KF4a ACCATGG.AC3 ACAAGG.ACGACGATGACAAGTTCTCGGAGGAGCTCAAGATCGGGGAG
KF4b CTCCCCG ATCTTG AGCTCCTCCG AG AACTTGTC ATCGTCGTCCTTGT AGTCC ATGGT
T a b le 3-2: P rim ers U sed to C lon e th e IR AK -1 V arian ts are Show n. Primers were purchased from A B l technologies
or Genosys. The amino acid residues added to the [IRAK 1-N( 1-211)], [IR A K l-N K (l-5 3 2 )] and [IRAK 1-K (212-532)]
constructs sequentially by site-directed mutagenesis PCR to create the FLAG-tag (D Y K D D D D K ) are highlighted in
3.3.3 Generation o f the [IRAK1-FL(1-712)], [IRAK1-N(1-211)], [IRAKI-
NK(l-532)] and the [IRAK1-NC(1-211:533-712)] constructs.
Full-length IRAKI, [IRAK 1 -FL( 1 -712)] was digested out of the pcDNA3 vector and the purified 2200bp fi-agment inserted into the pBKRSV vector using T4 DNA ligase (Section 2.2.8). Using the fiill-length wild-type IRAK-1 /pBKRSV plasmid as template, expression plasmids for the [IRAK1-N(1-21I)] and [IRAKI-NK( 1-532)] constructs were generated by introducing stop coAons!Kpnl sites (TGA) into the IRAK coding regicm immediately after the N-terminal region or kinase domains respectively. This was done using site-directed mutageneis using the primers N1/N2 and NK1/NK2 respectively (Section 2.2.11, table 2-1). The 3’ untranslated region of [IRAK1-N(1-211)] was removed by Kpnl restriction digestion, followed by religation. In order to clone the [IRAK1-NC(1-211:533-712)] construct, which lacked the entire kinase domain of IRAK-1, EcoRV sites were incorporated at each end of the kinase domain by site- directed mutagenesis using the primers NCla/NClb and NC2a/NC2b respectively (Section 2.2.11, Table 3- 2). EcoRV restriction enzyme digestion allowed the kinase domain to be removed and the remaining plasmid was religated to generate the fusion protein.
At GlaxoWellcome, two antibodies were raised against peptides located within the N-trnninal and C- terminal regions of IRAK-1. Unfortunately, only the rabbit IRAK-1 antibody raised against a peptide corresponding to amino acids 698-712 of human IRAK-1 proved to be usefiil. To permit detection of expression of constructs lacking this region, the [IRAKI-N( 1-211)], [IRAK 1-NK(1-532)] and [IRAKl- K(212-532)] constructs were NH
2
-terminally epitope tagged with the FLAG-peptide tag (DYKDDDDK). Nucleotide sequence encoding the FLAG-tag was inserted using QuikChange site-directed mutagenesis, according to the manufecturer’s protocol (Section 2.2. II). Four rounds of PCR were carried out in total for each construct, incorporating 2 amino acid residues of the FLAG-tag sequentially during each PCR reaction (shown in red in Table 3-1). To FLAG-tag the [IRAK1-N(1-211)] and [IRAK 1-NK(1-532)] constructs, primers NFla-NF4a were used alongside their complementary reverse primers, NFlb-NF4b (see Table 3-2) and primers KF1-KF4 were used in the case of the [IRAK 1-K(212-532)] clone. All the IRAK-1 variants were sequenced by the GlaxoWellcome DNA Sequencing Facility (Section 2.2.9).Once sequencing had verified that the IRAK-1 variants were correctly cloned, the DNA was isolated using Qiagen Maxiprep kits (Section 2.2.1.2), followed by caesium chloride purification of the plasmid DNA (Sections 2.2.2), This method of DNA extraction produced DNA of sufficient purity to give good transfection efficiencies and consequently allowed adequate protein expression to detect by western blot analysis (see Section 2.8.7).