COMENTARIO DEL TESISTA:

In order to better understand the structurally and functionally elusive DRG-DFRP protein complexes, the yeast Drg1-Dfrp1/Drg2-Dfrp2 proteins (Rbg1-Tma46, Rbg2-Gir2 respectively) and the human Drg1-Dfrp1 (Drg1-Lerepo4) proteins were expressed and purified for subsequent crystallization trials and GTP/RNA binding assays. The various constructs are given in table 3.1. All constructs of Rbg1 with Tma46 and Rbg2 with Gir2 were obtained from Dr. Bertrand Séraphin as part of the 3D Repertoire project. The constructs of Drg1 and Lerepo4 were made respectively by Mercedes Spinola and Isabel Perez in the laboratory.

The different proteins were essentially overexpressed in E.coli expression system using the autoinduction method unless otherwise specified (Refer section 3.1.2, table 3.3). All individual proteins had a 6-histidine tag at the N-terminal except when expressed along with its binding partner in which case only one of them carried the His tag (as given in table 3.1). The individual or complex proteins were isolated from the cell lysate by chelating affinity column chromatography loaded with nickel, by virtue of the presence of the 6-His tag which binds to the nickel ions in the column resin. Upon applying a gradient of 20 to 500 mM Imidazole which competes with the histidines for nickel binding sites, the proteins generally eluted out at around 50-100 mM of Imidazole concentration in the buffer.

All of the proteins could be efficiently extracted from the bacterial lysate in this manner demonstrating that the His tags at the N-terminal were accessible. The protein containing fractions from the nickel affinity chromatography as visualized in a polyacrylamide gel (SDS-PAGE) were concentrated and purified using a size exclusion column to achieve structurally homogenous, crystallization-grade, pure protein.

The DRG-DFRP protein complexes always eluted as a single heterodimer in size exclusion chromatography. Apart from being a strong binding partner, DFRP proteins have been thought to be necessary for the functional and physical existence of the DRG proteins (Ishikawa et al., 2005). Consistent with this observation, although Rbg1fl and Drg1fl could

be overexpressed individually in bacteria and purified as shown in lanes 4 and 7 respectively in figure 4.1.C, Rbg2fl could not be expressed alone. However, when

cotransformed with Gir2fl, the Rbg2fl-Gir2fl complex could be obtained which suggests that

Rbg2 could not have been expressed or degraded upon expression in the absence of Gir2fl.

Full length Tma46 on the other hand, could not be overexpressed in E. coli and degrades when expressed even as a complex with Rbg1. However, several stable C-terminal truncations of Tma46 in complex with Rbg1 could be expressed and purified as given for example in lanes 1-3 in figure 4.1.C.

Figure 4.1: Purification results for the DRG-DFRP complexes.

A. Chromatogram for the first purification step of the Rbg1fl-Tma46205-345 complex using the nickel chelating

HisTrap fast flow column. The peaks correspond to the elution of the protein given by the UV absorption at 280 nm (blue line). The increasing percentage concentration of buffer B (containing 500 mM Imidazole) during the gradient is given as the green line (0-100%). The fractions 5, 6 and 7 corresponding to the peak (25.5% B) was found to contain the Rbg1fl-Tma46205-345 complex as visualized in the Coomassie-stained

SDS-PAGE gel and was concentrated.

B. The concentrated Rbg1fl-Tma46205-345 protein sample was then loaded in a Superdex 200 26/60 column.

The size exclusion chromatogram shows the elution of the protein at 190.15 ml which corresponds to a molecular weight of ~70 kDa (MW of complex is 57.5 kDa and so suggests a single heterodimer). The fractions 37-40 were concentrated and stored at -80ºC.

C. The concentrated samples after purification by affinity followed by size exclusion chromatography techniques of the proteins Rbg1fl-Tma46205-345 (as representative), Rbg1fl-Tma46154-345, Rbg1fl-Tma46137-345,

Rbg1fl, Rbg2fl-Gir2174-238, Gir2fl, Drg1fl, Lerepo4220-426 and Drg1fl-Lerepo4220-426 is given. The latter Drg1fl-

Lerepo4220-426 complex was obtained by mixing together the individually expressed and affinity purified

Drg1fl and Lerepo4220-426 proteins and loading in the size exclusion column and separately concentrating the

fractions containing the complex. The marker used for the protein gel was Precision plus Protein Dual Color Standards (BioRad).

Although Rbg1fl-Tma46137-345, Rbg1fl-Tma46154-345 and Rbg1fl-Tma46205-345 were all

used for the crystallization trials, only the complex Rbg1fl-Tma46205-345 could be

successfully crystallized. The representative purification result for the Rbg1fl-Tma46205-345

construct is given in figure 4.1 with the chromatograms from the nickel chelating affinity (4.1.A) and size exclusion (4.1.B) chromatographic methods as all the other proteins were purified following the same protocol. Representative samples of the final purified proteins run on an SDS-PAGE gel is as shown in figure 4.1.C. Human Lerepo4 full length could neither be expressed in bacteria, but the C-terminal (220-396) could be obtained as shown in lane 8. The N-terminal Lerepo41-220 protein was obtained from Isabel Perez in the

laboratory. Most of the expressed proteins, especially complexes, were highly soluble and could be concentrated to high concentrations even as much as 180 mg/ml. Exceptions were Rbg1 and Drg1 which had to be concentrated to much lower levels (3-10 mg/ml) in order to avoid precipitation.

As previously reported, Gir2fl protein migrated at a size of around 70 kDa in an

SDS-PAGE gel although the molecular weight of the protein is 31 kDa (lane 6). This was due to the anomalous electrophoretic behavior contributed mainly by its highly acidic N- terminal RWD domain (Alves and Castilho 2005, Alves et al., 2004). The Rbg2 full length protein in complex with C-terminal dfrp fragment of Gir2 (Rbg2fl-Gir2174-238) was also