GOMEZ Gabriela Elena
congresos y reuniones científicas
An improved method for the purification of human alpha synuclein
La Plata. Buenos Aires
Congreso; XLVII Reunión Anual de la Sociedad Argentina de Biofísica; 2018
Human α-synuclein (AS) is a 140-residue, natively unfolded protein that can be an interesting study model for a protein surface labeling technique developed in our laboratory. Thus, it is important to obtain the protein with a high degree of purity. AS was expressed in Escherichia coli using plasmid pT7-7 (courtesy of Soledad Celej, CIQUIBIC, UNC-CONICET). Following transformation, BL21 electrocompetent cells were grown in LB medium with ampicillin (100 μg/ml). At OD600 nm ~ 0.70, cells were induced with 1 mM IPTG, cultured 4 h at 37 °C and harvested by centrifugation (Sorvall® Instruments). The cell pellet was resuspended in lysis buffer (50 mM Tris-HCl pH=7.4, 5 mM EDTA, 0.5% Tritón X-100, 1 mM PMSF and lysozyme) and incubated 1 h at 37 °C. This suspension was sonicated in ice bath with pulses of 10 seg in a Branson Ultrasonics Sonifier. Thereafter, MgCl2 (final concentration = 20mM) and DNAse (Sigma) were added and incubated 1 h at room temperature. Excess metal was removed with EDTA (final concentration = 20mM) and cellular debris was removed by centrifugation. The supernatant was boiled for 20 minutes and centrifuged. AS remained in this new supernatant which was exhaustive dialysed. The solution was loaded onto a pre-packed HiPrep 16/10 Phenyl FF (high sub) hydrophobic interaction column (GE Healthcare) and washed with 2 M ammonium sulfate in 20 mM K phosphate buffer (pH = 7.4), 1mM EGTA. AS eluted with decreasing salt and was exhaustive dialysed again. The protein solution was concentrated using a 10 kDa Amicon® Ultra-15 centifugal filter device (Millipore) and loaded onto a HiLoad 16/60 Superdex® 200 prep grade gel filtration column (Pharmacia Biotech). Size exclusion chromatography was run in 20 mM K phosphate buffer (pH = 7.4), 1mM EGTA, 50 mM NaCl. Protein concentration was estimated from the absorbance at 275 nm using an extinction coefficient of 5600 M−1 cm−1. Protein mass was confirmed by ESI mass spectrometry (Thermo Finnigan LCQ duo) and CD spectra (Jasco J-810 spectropolarimeter) at pH 4 or 7 were performed to evaluate AS structural integrity. The experimental protocol presented here was useful to express and purify functional AS without the presence of proteolytic degradation fragments.