Investigation of the binding affinity of Gata-1:Lmo1 Binding Affinity

A wider aim in the laboratory was to determine structures of components of the transcriptional complex. To that end, a tethered construct containing Ldb1LID-Lmo2LIM2-Gata-1NF was

expressed and purified. Unfortunately, NMR analysis indicated that the residues at the Lmo2:Gata1 interface were in intermediate exchange, which hampered structure determination. As such, other Lmo proteins were tested to establish if they could interact with Gata-1NF. GST-pull-down experiments were used to show that Lmo1-Ldb1LID, Lmo2-Ldb1LID

Chapter 3. Investigation of interactions within the Tal1/E2a/Lmo/Ldb/Gata pentameric complex 51

Lmo4-Ldb1LID did not [122]. As a result, Lmo1 was selected for further study because it can

also induce T-ALL and is generally considered to substitute for Lmo2 in Gata-1-containing transcriptional complexes [122]. In this chapter experiments are described to further characterise the Lmo1:Gata-1NF interaction by quantification of the binding affinity.

3.2.1 Expression and purification of constructs Gata-1NF

Gata-1NF, the N-terminal finger of Gata-1 (comprising residues 200–254 of the mouse protein)

was expressed using 0.4 mM IPTG induction in BL21(DE3) E. coli cells and purified by glutathione (GSH) affinity chromatography and treatment with thrombin to remove the GST tag, prior to cation exchange chromatography, as described in section 2.3 (Figure 3.2). The largely soluble GST-Gata1NF protein was purified by GST-chromatography (Figure 3.2A) and

each elution fraction from the GSH resin was separately treated with thrombin resulting in ~50% removal of the GST-tag as judged by SDS-PAGE (Figure 3.2B). Gata-1NF was not fully

separated from GST and uncleaved GST-Gata-1NF after cation exchange chromatography

(Figure 3.2C). Thus, the samples were submitted to size-exclusion chromatography (Figure 3.2D). This yielded approximately 5 mL of 50 µM Gata-1NF from 3 L of expression culture.

Even following size-exclusion chromatography fractions still contained small amounts of uncleaved GST-Gata-1NF protein as seen by SDS-PAGE.

Chapter 3. Investigation of interactions within the Tal1/E2a/Lmo/Ldb/Gata pentameric complex 52

Figure 3.2 SDS-PAGE analysis of Gata-1NF purification. Gata-1NF expressed in BL21(DE3) cells

grown at 37 °C before 0.4 mM IPTG induction at OD600 nm ~0.6 followed by A. GSH-affinity

purification. Soluble lysate was incubated on GSH beads, followed by washing and elution with 20 mM glutathione in 20 mM Tris, 150 mM NaCl, 3 mM CaCl2, 0.1% v/v 2-mercaptoethanol, pH 7.4. B.

Thrombin cleavage. Each fraction of GST-Gata-1NF was treated overnight with 200 U thrombin to

remove the GST-tag. C. Cation exchange chromatography. All fractions of cleaved protein were pooled and subjected to cation exchange chromatography using a UnoS column in 20 mM Tris, 1 mM DTT,

Chapter 3. Investigation of interactions within the Tal1/E2a/Lmo/Ldb/Gata pentameric complex 53

pH 7.4, with a 0–1 M NaCl gradient, with fractions collected from 300–600 mM NaCl. D. Size- exclusion chromatography. Fractions from cation exchange chromatography were pooled and concentrated prior to being subjected to size exclusion chromatography on a S75 column in 20 mM Tris, 100 mM NaCl, 1 mM TCEP, pH 7.

Lmo1-Ldb1LID

Lmo1 is insoluble and prone to aggregation, and thus was tethered to Ldb1LID for expression

[56, 57]. The Lmo1-Ldb1LID construct used for this assay contained both LIM domains (LIM1

and LIM2) of Lmo1 and the full Ldb1LID (residues 295–339). The fusion construct was

expressed and purified as described in section 2.3 (Figure 3.3). The construct was expressed in BL21(DE3) E. coli cells using 0.4 mM IPTG induction, followed by GSH affinity chromatography and on bead thrombin cleavage (Figure 3.3A). Protein was further purified by size exclusion chromatography, where it eluted in two peaks of high purity. Fractions from each peak were pooled together and kept for further analysis (20 mL of 41 µM Lmo1-Ldb1LID

Chapter 3. Investigation of interactions within the Tal1/E2a/Lmo/Ldb/Gata pentameric complex 54

Figure 3.3 SDS-PAGE analysis of Lmo1-Ldb1LID purification. BL21(DE3) cells were grown at 37

°C before induction with 0.4 mM IPTG at OD600 nm ~ 0.6 for 22 h at 22 °C. A. GSH-Affinity

purification. Soluble cell lysate was applied to GSH resin, followed by a wash step and cleavage with thrombin (200 U) in 20 mM Tris, 150 mM NaCl, 3 mM CaCl2, 0.1% v/v 2-mercaptoethanol, pH 8 to

release the untagged protein. B. Size exclusion chromatography. Cleaved protein was concentrated and run on S75 column in 20 mM Tris, 100 mM NaCl, 1 mM TCEP, pH 7.

3.2.2 Binding of Gata-1NF to Lmo1-Ldb1LID

Isothermal titration calorimetry (ITC) was used to determine binding affinity of Gata-1NF for

Lmo1-Ldb1LID (Figure 3.4) as described in section 2.6. Gata-1NF was separately titrated into

two slightly different concentrations of Lmo1-Ldb1LID,both from the same batch of purified

protein. The signal to noise in the experiment was relatively low, suggesting a low enthalpy under the conditions of the experiment. When Lmo1-Ldb1LID was used at 26 µM, it was noted

Chapter 3. Investigation of interactions within the Tal1/E2a/Lmo/Ldb/Gata pentameric complex 55

that the stoichiometric ratio was lower than expected (0.5:1, rather than 1:1) (Table 3.2), which likely arises from the low signal-to-noise and/or errors in estimation of protein concentrations. When using a 19 µM solution, the stochiometric ratio was 1.1:1. Binding affinity is less sensitive to errors in protein concentration than stoichiometry, so those values from the two experiments were consistent, giving rise to an average binding affinity (KA) of ~2 × 105 M-1.

Figure 3.4 ITC binding data for Gata-1NF titrated into Lmo1-Ldb1LID. Gata-1NF (175 µM) was

titrated into Lmo1-Ldb1LID at A. 26 µM and B. 19 µM. Experiments were carried out in 20 mM MES,

100 mM NaCl, 1 mM TCEP, pH 6.5 at 25 °C.

TABLE 3.2 ITC data. The association constant (KA) and the stoichiometric ratio (n) were calculated

at two different concentrations of Lmo1-Ldb1LID as indicated.

Parameter 26 µM Lmo1-Ldb1LID 19 µM Lmo1-Ldb1LID

KA (M-1) 1.2 ± 0.3 × 105 3.3 ± 0.9 × 105

n 0.50 ± 0.06 1.10 ± 0.03