Características de la Propuesta Planteada

Various substituted ubiquitins and Ubc1K93R (hence forward just Ubc1) from S. cerevisiae were over expressed in BL21(DE3)pLysS E.coli strain and purified using a Q anion exchange column, followed by a Sephadex G75 (GE Healthcare) size exclusion column as previously described (11). Electrospray ionization mass spectrometry confirmed the integrity of Ubc1 (MWobs 24162.4 ± 0.8Da, MWcalc 24162.2). Kathryn R. Barber purified Ubc1. Protein concentrations of Ubc1 were determined through Bradford (BioRad) reactions performed in triplicate. Protein concentrations of Ub were determined by weight of lyophilized proteins.

Hexahistidine (His6_{) tagged HIP2}C170S _{(hence forward just HIP2) was} overexpressed in the BL21(DE3)star E. coli strain. The bacteria were grown at 37 °C

overnight in LB media (10 mL) containing the antibiotic kanamycin (30 µg / mL). The

culture was then diluted 1:100 into 1 L of LB containing the same antibiotics and grown at 37 °C to an A600 between 0.60-0.75. Protein expression was induced for 16-20 hours with 0.7 mM IPTG at 15 °C. Harvested cells were re-suspended in 20 mM Tris-HCl, 200

mM NaCl, 1 mM TCEP, 5 mM imidazole at pH 8.0 with the addition of a COMPLETE mini EDTA-free protease inhibitor cocktail tablet (Roche Diagnostics). Cells were lysed with a French pressure cell and cellular debris was removed by centrifugation at 95500 xg. All following purification procedures were performed at 4 °C to minimize protein

degradation. The clarified extract containing His6-HIP2 was applied to Ni-NTA Agarose (Qiagen) and washed with 20 mM Tris-HCl, 200 mM NaCl, 1 mM TCEP, 10 mM imidazole at pH 8.0 to elute non specifically bound material. His6-HIP2 was eluted with 20 mM Tris-HCl, 200 mM NaCl, 1mM TCEP, 200 mM imidazole pH 8.0. The protein was concentrated and loaded on to a Superdex 75 10/300 GL column (GE Healthcare) in 20 mM Tris-HCl, 200 mM NaCl, 1mM TCEP, 10 mM imidazole pH 8.0. Thrombin (2

µg / mL) was utilized to cleave the His6 tag at room temperature for two hours and a

subsequent Ni-NTA Agarose column was utilized to remove the tag and any uncut protein from HIP2. A final Superdex 75 10/300 GL column run in 20 mM Tris-HCl, 200 mM NaCl, 1 mM TCEP pH 8.0 was used to remove residual thrombin from the purified HIP2 protein. Purification steps were monitored using SDS-PAGE. SDS-PAGE was performed using 16.5% tris-tricine gels in a mini-gel system (BioRad) and was stained with Coomassie dye. HIP2 concentrations were determined through Bradford (BioRad) reactions performed in triplicate. Purified HIP2 was confirmed using electrospray ionization mass spectrometry (MWobs 22534.8 ± 0.3Da, MWcalc 22534.7).

2.2.3Protein expression and purification of NCys-Ub and NCys-UbK48R

Human Ub proteins were modified to contain a cysteine residue in a peptide extension (GPCLGS) at the N-terminus of both wild type Ub (NCys-Ub) and UbK48R

(NCys-UbK48R). GST fusion proteins of NCys-Ub and NCys-UbK48R were over expressed in the BL21(DE3)pLysS E.coli strain. Cell cultures were grown overnight in LB media (10 mL) containing the antibiotic carbenicillin (50 µg / mL). The culture was then diluted

1:100 in LB media containing the same antibiotic and grown at 37 °C to an A600 of 0.60. Protein expression was induced with 0.5 mM IPTG for 4 hours at 37 °C. Harvested cells

were re-suspended in binding buffer consisting of 25 mM Tris-HCl, 100 mM NaCl, 3 mM DTT at pH 7.5 with the addition of a COMPLETE mini EDTA-free protease inhibitor cocktail tablet (Roche Diagnostics). Cells were lysed with a French pressure cell and cellular debris was removed by centrifugation at 95500 xg. The lysate was purified using a GST affinity column (GE Biosciences), washed with the same binding buffer and eluted with 25 mM Tris-HCl, 100 mM NaCl, 3 mM DTT, and 20 mM glutathione. The GST affinity tag was cut using TEV (Shaw lab; 50 µg / mL) and

removal of the GST tag, any uncut protein and the TEV protease was achieved by size exclusion chromatography on a Sephadex G-75 column (GE Healthcare) with 25 mM Tris-HCl, 100 mM NaCl, 0.5 mM EDTA pH 7.4. Purification steps were monitored using SDS-PAGE. SDS-PAGE was performed using 16.5% tris-tricine gels in a mini-gel system (BioRad) and was stained with Coomassie dye. NCys-Ub and NCys-UbK48R concentrations were determined through SDS-PAGE comparison to Ub substitutions. Purified NCys-Ub and NCys-UbK48R were confirmed using electrospray ionization mass spectrometry (NCys-UbK48R: MWobs 9107.61 ± 0.42 MWcalc 9107.4; NCys-Ub: MWobs 9079.08 ± 0.94 MWcalc 9079.44).

NCys-Ub and NCys-UbK48R were then reacted with Alexa-Fluor 680 C2-Maleimide (Invitrogen) to fluorescently label the Ub and UbK48R. The cysteine in NCys-Ub and NCys-

UbK48R reacts with the C2-maleimide to yield Alexa-Ub and Alexa-UbK48R. These reactions were performed by a 10:1 (v/v) mixing of 100 µM NCys-Ub in 100 mM NaCl,

0.5 mM EDTA, 25 mM Tris-HCl pH 7.4 and 5 mM Alexa-Flour in 100% isopropanol respectively. This reaction was stopped with the addition of 5 mM DTT, and then purified using a PD-10 desalting column (GE Healthcare) and extensive dialysis with 25 mM Tris-HCl, 100 mM NaCl pH 7.3 was used to remove unreacted Alexa dye. Ub molecules were confirmed to be labelled with Alexa via electrospray ionization mass spectrometry (Alexa-UbK48R _MW

obs 10089.21 ± 0.31 agrees with expected values for NCys-UbK48R (9107.46 Da) and Alexa dye (~982 Da)); Alexa-Ub MWobs 10061.89 ± 0.40 agrees with expected values for NCys-Ub (9079.44 Da) and Alexa dye (~982 Da)).