Study sheds light on how scramblase proteins rearrange cell membranes cornell NatureComms
. For wiltype and mutant afTMEM16 the leak is >2 orders of magnitude smaller than the rate constant of protein-mediated scrambling and therefore is negligible. All conditions were tested side by side with a control preparation of WT afTMEM16 reconstituted in C18 lipids. In some rare cases this control sample behaved anomalously, judged by scrambling fit parameters outside 3 times the standard deviation of the mean for the wildtype.
. Briefly, MSP1E3 in a pET vector was transformed into the BL21-Gold strain . Transformed cells were grown in LB media supplemented with Kanamycin to an ODof 0.8 and expression was induced with 1 mM IPTG for 3 h. Cells were harvested and resuspended in 40 mM Tris-HCl pH 78.0, 300 mM NaCl supplemented with 1% Triton X-100, 5 μg mlpepstatin, 100 μM phenylmethane sulphonylfluoride and protease inhibitor cocktail tablets .
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