(Figure 8, D, G, K). The Resource-S column, a strong cation exchanger, was chosen based on the fusion constructs’ pI and pH of buffer such that only ID2 bound to the column and not the tag. At this stage, there was still some residual tag left. To avoid protein loss, no gel filtration chromatography was performed. Instead, a final round of reverse His-affinity chromatography (Figure 8E) was run to remove the residual tag. Running at a slow gradient of increasing Imidazole concentration, the His6-containing tag was captured on the nickel beads while pure ID2 was eluted in the flowthrough (Figure 8, F, H). The seleno-methionine construct was pure enough after ion exchange chromatography and did not require further purification. The proteins were pooled and concentrated in buffer 50 mM TRIS-HCL pH8.0, 100mM NaCl after the final chromatography step. 90% purity and greater was checked by SDS-PAGE (Figure 9) and gel filtration elution profiles showed that the protein existed as dimers in solution (Appendix 6). These samples were then used for crystallization experiments. ! 36! Figure 8: ID2 proteins’ expression and purification (A) Elution profile of nickel-bead affinity and desalting chromatography. (B) SDS-PAGE: marker (lane 1), sample before induction (lane 2), pooled desalted fractions (lane 3), TEV-cleaved affinity tag (lane 4). (C) Elution profile of ion-exchange chromatography with increasing salt gradient. (D) SDS-PAGE: marker (lane 1), input sample after TEV (lane 2), flowthrough (lane 3), fractions at positions of crosses in profile from Fig 3.1C (lane 4-11). (E) Reverse affinity chromatography profile; ID2 eluted in flowthrough, residual tag bound to nickel beads. (F) SDS-PAGE: Representative ID2 fractions (lane 1-2), marker (lane 3), affinity tag (lane 4) ! 37! Figure continued: (G) N-HLH82-L expression and purification SDS-PAGE: marker (lane 1), pooled affinity and desalting chromatography fractions before TEV (lane 2), TEV cleaved (lane 3), unbound ion exchange fraction (lane 4), ion exchange elutions (lane 5-10). (H) N-HLH-82-L reverse affinity chromatography SDS-PAGE: input sample (lane 1), marker (lane 2), bound tag (lane 3), unbound ID2 fractions (lane 4-12). (J) HLH24-82-L-Se-Met SDS-PAGE: marker (lane 1), sample before induction (lane 2), pooled affinity and desalting fractions before TEV (lane 3), TEV cleaved (lane 4). (K) HLH24-82-L-Se-Met ion exchange samples SDS-PAGE: input sample after TEV (lane 1), marker (lane 2), eluted bound ID2 fractions (lane 3-8). Figure 9: ID2 proteins’ purity check by SDS_PAGE: marker (lane M, kDa). N-HLH82-L (gel A, lane 1), HLH24-82-L (gel B, lane 2), HLH24-82-L-Se-Met (gel C, lane 3) ! 38! 3.3 Protein Identification The purified samples were excised from the gel and analyzed by Liquid chromatography tandem mass spectrometry (LC/MS/MS) for peptide mass fingerprinting. Searches were made both against all non-redundant proteins as well as just the human dataset to show that no matter which dataset was used, the results were the same and the identity of the protein was that of ID2 (Table 7). The only difference was that the longer form of ID2, N-HLH82-L contained the intact N-terminal region of the protein as shown in matched peptides in bold red. Table 7: LC/MS/MS mass spectrometry top hits for the purified proteins (Figure 8). Searches were done against all nr as well as human nr to show that the fragments captured always belonged to ID2. Note that the N-HLH-82-L contained the intact N-terminus (matched peptides in bold red) whereas the shorter form HLH24-82-L and the seleno-methionine version did not. N-HLH82-L (searched against all nonredundant proteins) HLH24-82-L (searched only in human protein dataset) HLH24-82-LSe-Met (searched against all nonredundant proteins with Se variable modification set) ! Match to: IPI00294210 Score: 4673 Tax_Id=9606 Gene_Symbol=ID2 DNAbinding protein inhibitor ID-2 Found in search of C:\Documents and Settings\Administrator\Desktop\230609_p565C6_pure.RAW Nominal mass (Mr): 15022; Calculated pI value: 7.82 NCBI BLAST search of IPI00294210 against nr Unformatted sequence string for pasting into other applications Fixed modifications: Carbamidomethyl (C) Variable modifications: Oxidation (M) Cleavage by Trypsin: cuts C-term side of KR unless next residue is P Sequence Coverage: 42% Matched peptides shown in Bold Red MKAFSPVRSV RKNSLSDHSL GISRSKTPVD DPMSLLYNMN DCYSKLKELV 51 PSIPQNKKVS KMEILQHVID YILDLQIALD SHPTIVSLHH QRPGQNQASR 101 TPLTTLNTDI SILSLQASEF PSELMSNDSK ALCG Match to: Q53H99_HUMAN Score: 17248 Inhibitor of DNA binding variant (Fragment).- Homo sapiens (Human). Found in search of C:\Documents and Settings\Administrator\Desktop\Marie\1D2_BHLH.RAW Nominal mass (Mr): 15071; Calculated pI value: 7.82 NCBI BLAST search of Q53H99_HUMAN against nr Unformatted sequence string for pasting into other applications Taxonomy: Homo sapiens Links to retrieve other entries containing this sequence from NCBI Entrez: BAD96402 from Homo sapiens Fixed modifications: Carbamidomethyl (C) Variable modifications: Oxidation (M) Cleavage by Trypsin: cuts C-term side of KR unless next residue is P Sequence Coverage: 25% Matched peptides shown in Bold Red MKAFSPVRSV RKYSLSDHSL GISRSKTPVD DPMSLLYNMN DCYSKLKELV 51 PSIPQNKKVS KMEILQHVID YILDLQIALD SHPTIVSLHH QRPGQNQASR 101 TPLTTLNTDI SILSLQASEF PSELMSNDSK ALCG Match to: IPI00294210 Score: 1435 Tax_Id=9606 Gene_Symbol=ID2 DNA-binding protein inhibitor ID-2 Found in search of C:\Documents and Settings\Administrator\Desktop\Se_Met_C3_1.RAW Nominal mass (Mr): 15022; Calculated pI value: 7.82 NCBI BLAST search of IPI00294210 against nr Unformatted sequence string for pasting into other applications Fixed modifications: Carbamidomethyl (C) Variable modifications: Oxidation (M),Delta:S (-1)Se (1) (M) Cleavage by Trypsin: cuts C-term side of KR unless next residue is P Sequence Coverage: 25% Matched peptides shown in Bold Red MKAFSPVRSV RKNSLSDHSL GISRSKTPVD DPMSLLYNMN DCYSKLKELV 51 PSIPQNKKVS KMEILQHVID YILDLQIALD SHPTIVSLHH QRPGQNQASR 101 TPLTTLNTDI SILSLQASEF PSELMSNDSK ALCG 39! 3.4 Crystallization Crystal screens were done initially in a high-throughput 96-well sitting-drop format using an Innovadyne robot to dispense the screening solutions and protein at 200 nl volumes. This enabled many different screens from both Qiagen and Hampton Research to be tested. HLH24-82-L was screened and the best hit was found with the Cation Suite from Qiagen. In the condition 0.1 M MES pH 6.5, 2.5 M Lithium Acetate, crystals were small, needle like and tended to clump together. To optimize growth, manual hanging-drop screens were setup in a larger final volume of µl to allow more space for growth. At 18°C, the crystals were still quite small and clumpy (Figure 10A). There was no significant difference in the size of crystals grown at different temperatures but those at 18°C had the sharpest edges so this temperature was maintained for further optimization steps. To try obtaining a single crystal that had a large enough needle that could be broken off for mounting, microseeding technique was used to aid in nucleation. The crystals in Figure 10A were crushed and serial dilutions made from the stock solution were then used to setup the drops of µl of the diluted mixture with µl of precipitant solution and µl protein solution. With increasing dilutions, there were fewer crystals, but they were larger and had fewer needles (Figure 10, B-D). Single needles were carefully broken off with a cryoloop and flash cooled in liquid nitrogen for X-ray diffraction studies. ! 40! . (searched only in human protein dataset) Match to: Q53H99_HUMAN Score: 17248 Inhibitor of DNA binding 2 variant (Fragment) Homo sapiens (Human). Found in search of C:Documents and SettingsAdministratorDesktopMarie1D2_BHLH.RAW. (lane M, kDa). N-HLH82-L (gel A, lane 1), HLH24-82-L (gel B, lane 2), HLH24-82-L-Se-Met (gel C, lane 3) ! 39 ! 3. 3 Protein Identification The purified samples were excised from the gel and. ID2 fractions (lane 1-2), marker (lane 3) , affinity tag (lane 4) ! 38 ! Figure 8 continued: (G) N-HLH82-L expression and purification SDS-PAGE: marker (lane 1), pooled affinity and desalting