The document discusses preliminary experiments to induce soluble rREL1 protein for use in high-throughput screening of small molecule compounds. It describes investigating the potency of 6 established lead molecules on cell viability and generating dose response data for Mordant Black 25. Conditions for expressing and purifying rREL1 under different lysis methods are explored. Plans are outlined for establishing a fluorescence-based ligase assay using labeled RNA fragments and optimizing various assay parameters.

1. Continuing with preliminary experiments to induce soluble rREL 1Continuing with preliminary experiments to induce soluble rREL 1
(for high throughput (FRET based) screening of small compounds)(for high throughput (FRET based) screening of small compounds)
Investigated actual potency of 6 established lead molecules with respectInvestigated actual potency of 6 established lead molecules with respect
to BS #427 Viability by coulter counting and Alamar blue assayto BS #427 Viability by coulter counting and Alamar blue assay
Large scale induction & purification of rREL 1Large scale induction & purification of rREL 1
Generate ECGenerate EC5050 data fordata for Mordant black 25Mordant black 25
Expressed & purified rREL 1 under different chemical lysis conditionsExpressed & purified rREL 1 under different chemical lysis conditions
and by paramagnetic beads respectivelyand by paramagnetic beads respectively

2. A fluorescence-based high-throughput ligase assay
Donor (D) = FAM, Acceptor (A) = Cy5, 250 nM
Assay will utilise a 104bp RNA fragment
(CYb) in concert with labelled 20mers ??
Coupling efficiency varies with fluorophore distance
70-100A is where FRET is achieved
Forster distance = Separation at which 50% photon transfer achieved
Mix & Measure AssayMix & Measure Assay
Compatible with 96 well plateCompatible with 96 well plate

3. Assay EstablishmentAssay Establishment
1)1) Purify catalytic domain (L1Purify catalytic domain (L151-32451-324
) of REL 1 (in association with TEV cleavable HisTag A2)) of REL 1 (in association with TEV cleavable HisTag A2)
by FPLC utilising NTA columns in concert with imidazole gradientby FPLC utilising NTA columns in concert with imidazole gradient
2) Express 104bp RNA (CYb) species from linear pBlueScript clone by In Vitro transcription
3) Anneal labelled 20mers
4) Test E Coli purified REL1 batch versus adenylation verified leads
5) Independently verify FRET by radioactive ligation assays
Assay OptimisationAssay Optimisation
Various parameters associated with ‘Mix & measure’ assay will be optimised, e.g.
 Gap distance: 16 donor/acceptor fluorophore combinations assayed over 0-12bp
hiatus
 RNA Substrate: Is CYb best ?
 Fluorophore species: TAMRA, Cy3, Cy5 etc
 Dynamic detection/sensitivity range: Serial substrate dilutions versus detection
 DMSO Concentration: 0.1% - 10%
Assay ImplementationAssay Implementation
1) Screen DDU (University of Dundee) ‘small compound’ library set(s)
2) Initial primary screen @ 10uM + 1% DMSO
3) Compounds >/- 3 x SD from (collective) median inhibition will be deemed ‘active’
and subject to counter screens

5. #1#1 #2#2 #3#3 #4#4
50KD
40KD
30KD
20KD
PP PP PP P?P?SS PP PP PP P?P?SS SS SS SS SS S?S? S?S?
+ + + +- - - -
Catalytic domainCatalytic domain
REL1REL1
Specific and high level expression of L1 (catalytic REL 1 domain) in pellet
Western Blotting has revealed some soluble L1 in induced supernatant
Is this due to a lack of A2 (N-terminal TEV His tag) rendering L1 soluble ?
+/- 0.5mM IPTG+/- 0.5mM IPTG
Western blotting
with:
 α REL 1Ab
 α his Tag Ab
A2A256-17656-176

6. ++ ++-- -- ++ ++-- --
Bug buster/PBS + lysozyme/
Benzonase + 1% Triton X-100
Buffer A + lysozyme/
Benzonase
+/- 0.5mM
IPTG
Buffer A + lysozyme/
Benzonase + 1% Triton
X-100
Bug buster/PBS + lysozyme/
Benzonase
Clone #1_2Clone #1_2 Clone #5_1Clone #5_1
PPPP PP PP PPSS SS
Clone #1_3Clone #1_3 Clone #5_2Clone #5_2
SS SSPP PP PP PPSSSS SS SS SS
Cultures grown to an initial OD600 of 0.5_0.6 at 37o
C for 1.5 hours by seeding 2ml of LB with 25ul of overnight
Induction achieved by splitting 2ml culture into 2 x 1ml aliquots and then adding 6.25ul of 1/10 0.8M IPTG to one such
aliquot (2nd
= un induced control) followed by growth @18o
C for 20-21 hours
Like original prep (i.e. Initial OD600 0.8 followed by induction for 20 hours @ 20o
C in presence of 0.5mM IPTG) = #1_1
REL 1 remains in pelleted fraction
Similarly, TEV cleavable N terminal His tagged A2 remains associated with pellet
5050
4040
3030
2020
1515
1010
3.53.5
L1
51-469
A2
56-176
 4-12% Bis Tris Novex gel
 1 x MES
 200V 45 minutes
Pellet
#1_1 (+)
Supernatant
#1_1(+)

7. Re transform BL21_DE3 with pET21d !Re transform BL21_DE3 with pET21d ! Monday
Set up overnight culturesSet up overnight cultures Tuesday
Set up orbital shaker for
2 litre flasks (x4) !
Set up 4 x 500ml IPTG inductions fromSet up 4 x 500ml IPTG inductions from
single clone in 4 x 2L flaskssingle clone in 4 x 2L flasks over 20 hoursover 20 hours
@ 20@ 20oo
CC
wednesday
Need orbital shaker
for 20 hours @ 20o
C !
Lyse frozen pellets in CTCB by mechanical lysisLyse frozen pellets in CTCB by mechanical lysis Thursday
Purify L1-A2 fraction by LC using an Imidazole gradientPurify L1-A2 fraction by LC using an Imidazole gradient Friday

8. Effect of 100uM solute (NCI compound) on BS # 427 cells versus DMSO (only)
solvent
162535
MordantBlack
45609
1698
125908
117079
0.4%DMSO
MediumOnly
0.00E+00
1.00E+06
2.00E+06
3.00E+06
4.00E+06
5.00E+06
6.00E+06
7.00E+06
8.00E+06
NCI Drug #
NumberofCells_Coultercounter
162535
Mordant Black
45609
1698
125908
117079
0.4% DMSO
Medium Only
z
99 % cell kill !99 % cell kill !