Aspectos Económicos.

The work in this part was done concurrently with experiments performed in Chapter 4 to sort out ΔNOTUB1 expression and purification problems. The UBE2D2 mutants which showed an interesting Y2H result were cloned into the pET151/D vector that fuses a His-tag to the protein and purified for NMR titration experiment with ΔNOTUB1, in case problems with production of ΔNOTUB1 could ever be resolved. Moreover, as an alternative to the pETM-11 vector, ΔNOTUB1 was also cloned into this vector to ensure that the problem of ΔNOTUB1 heterogeneity is not caused by pETM-11.

5.4.1 Directional TOPO® Cloning

The chosen method for cloning UBE2D2 mutants into suitable expression vector was to use the Champion TOPO® cloning kit from Invitrogen. The vector pET151/D contains the T7 bacteriophage promoter for a high inducible expression similar to pETM-11, and also has an N-terminal His-tag and TEV cleavage site (Figure 5.4A). TOPO® cloning does not require any DNA ligases to clone the targeted genes into specific vectors. This system merely relies on Taq polymerase to amplify the gene with primers containing TOPO® sites (CACC). The vital feature of this system is the pre-linearised vector containing topoisomerase at the 5’ and 3’ ends of the vector which functions as both restriction enzyme and ligase. Directional

TOPO® cloning enables cloning of the blunt-ended PCR products in a 5´ to 3´ orientation directly into the vector in 5 minutes ligation reaction, thereby eliminating subcloning steps and saving a lot of time. The vectors contain a single-strand GTGG overhang on the 5´ end and a blunt end on the 3´ end. The four-nucleotide overhang invades the double-strand DNA of the PCR product and anneals to the CACC sequence at 5´ end and topoisomerase will then ligate the PCR product in the correct orientation, similar to the method described in Shuman (1994).

Chapter Five | TARGETED ANALYSIS OF E2:OTUB1 COMPLEXES

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Figure 5.4 Map of pET151/D-TOPO® vector: Visible prior to the topoisomerase site are His-tag and TEV site, which function in producing the expressed protein with a cleavable N-terminal 6xHis-tag. Ampicillin resistance serves as a selectable marker for this vector.

Chapter Five | TARGETED ANALYSIS OF E2:OTUB1 COMPLEXES

Page | 159 5.4.1.1 Primer design and the TOPO® cloning principle

All forward primers have the CACC sequence at the 5’ end of the primer; this was to ensure the topoisomerase reaction worked. The remainder of the primer was designed to provide a suitable annealing length complementary to the two strands of the target DNA, with a melting temperature (Tm) between 50°C-58°C and the Tm difference between the forward and reverse primers should be no more than 2°C to allow efficient PCR reaction.

Table 5.2 TOPO® forward and reverse primers: ORF Primer 5’ to 3’ (first base position indicated at 5’ end) with CACC addition denoted by the green font.

Forward primer Reverse primer

OTUB1 5' CAC CAT GGC GGC GGA

GGA AC 3'

5' CTA TTT GTA GAG GAT ATC GTA GTG TCC 3'

ΔN

OTUB1 5' CAC CGA GAT TGC TGT

GCA GAA CC 3'

5' CTA TTT GTA GAG GAT ATC GTA GTG TCC 3'

UBE2D2 5' CAC CAT GGC TCT GAA

GAG AAT CCA C 3'

5' TTA CAT CGC ATA CTT CTG AGT CC 3'

UBE2E1 5' CAC CAT GTC GGA TGA

CGA TTC G 3' 5' TTATGTAGCGTATCTCTTGGTCC 3'

UBE2N 5' CAC CAT GGC CGG GCT G

3'

5' TTA AAT ATT ATT CAT GGC ATA TAG CCT CGT CCA TG 3'

The PCR products were then incubated with the linear vector that already has the topoisomerase enzyme covalently attached to both of its strands' free 3' ends. When the free 5' ends of the PCR product strands attack the topoisomerase (or 3' end) of each vector strand, the strands are covalently linked by the already bound topoisomerase. This reaction proceeds efficiently when this solution is incubated at room temperature with required salt to prevent re-cutting by the topoisomerase. Reaction products were transformed into chemically competent cells and positive transformants were selected from ampicillin selection plate. Overall, TOPO® cloning proved to be a more convenient protocol compared to conventional cloning with the pETM-11 vector.

Chapter Five | TARGETED ANALYSIS OF E2:OTUB1 COMPLEXES

Page | 160 5.4.1.2 Purification of UBE2D2 mutant proteins

Following cloning of the UBE2D2 mutants into the His-tag pET151/D TOPO® vector, they were transformed into Rosetta expression cells for large-scale protein purification. Protein expression and purification were performed in the same manner as for wild type UBE2D2. Although some modifications in ion exchange chromatography were required due to the pI being either increased or decreased upon amino acids substitution, all mutants were purified in satisfying yield and were found to be correctly folded by analysis of 1D NMR spectra. However, no binding experiments could be done on them until the end of this project since the problem with ΔNOTUB1 could not be solved.

Chapter Five | TARGETED ANALYSIS OF E2:OTUB1 COMPLEXES

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