Supplementary Figure S3 from Expression Defect Size among Unclassified <i>MLH1</i> Variants Determines Pathogenicity in Lynch Syndrome Diagnosis
Authors: Hinrichsen Inga , Brieger Angela , Trojan Jörg , Zeuzem Stefan , Nilbert Mef , Plotz Guido 
Published: April 2023

<p>PDF file - 75 KB, Proteolytic and thermal stability of MLH1 variants. A. Pulse-chase kinetics of MLH1 variants. MLH1 was co-expressed with PMS2 in vitro using pcDNA3-MLH1 and pSG5-PMS2 vectors in the presence of 35S-methionine (EasyTag Methionine, PerkinElmer) with the TNT T7 transcription/translation system (Promega) in 50 �l for 30 min at 30 {degree sign}C. After synthesis stop with 1.5 �g DNAse I, 1.5 �g RNAse A and 500 nmol methionine), samples were incubated at 37 {degree sign}C for up to 5 hours, and aliquots of 5 �l were removed after several time points. MLH1 protein concentration was analyzed by SDS-PAGE and phosphoimaging on an LAS9000 with MultiGauge (Fuji). Signals were normalized for t=0, and exponential regression lines were calculated. Half-life times were determined from the slopes of the regression lines. B. Thermal stability of MLH1 variants. Recombinant baculoviruses for the expression of wild-type His-MLH1 and His-MLH1 variants were produced using the Bac-to-Bac-system (Invitogen) with pFastBac HT-MLH1 (generated by restriction-ligation from pcDNA3-MLH1). Sf9 suspensions (100 ml) were pelleted after 48 h and resuspended in 3 pellet volumes buffer A (20 mM Hepes KOH pH 7.6, 5 mM MgCl2, 0.1 mM EDTA). After incubation on ice for 15 min, cells were lysed in a Dounce homogenizer (pestle tight). One volume buffer C (20 mM Hepes KOH pH 7.6, 5 mM MgCl2, 0.1 mM EDTA, 600 mM NaCl, 40% Glycerol) was added, mixed and the sample rotated 30 min at 4 {degree sign}C. The extract was centrifuged. For purification, cell lysate was diluted to 250 mM NaCl with 25 mM Hepes, 10% Glycerol, 5 mM MgCl2, 10 mM Imidazol and incubated with 1 ml Ni-NTA-Agarose (Qiagen) for 1 h at 4 {degree sign}C. The agarose was washed 6 times with 10 ml wash buffer 1 (25 mM Hepes-KOH, pH 7.6, 10 % Glycerol, 5 mM MgCl2, 300 mM NaCl, 10 mM Imidazol, 0.035 % Mercaptoethanol) and 4 times with wash buffer 2 (identical except for NaCl concentration: 100 mM). Bound protein was eluted with 0.3 ml wash buffer 2 with 250 mM Imidazol. Two additional, identical elutions using 0.6 ml were performed. Purity was assessed by SDS-PAGE and Coomassie staining. For assessing thermal stability, these His-MLH1 protein preparations were diluted with reaction buffer (final concentration: 10 mM Hepes pH 7.5, 150 mM NaCl, 1�l/ml SyproOrange, Invitrogen) to a concentration of 1.2 �M. Reactions were performed in 10 �l in a StepOnePlus (AppliedBiosciences). Temperature was increased from 10{degree sign}C to 75 {degree sign}C in 0.3 {degree sign}C increments. Signals were detected at 580 nm using TAMRA calibration. Results were analyzed using GraphPadPrism (GraphPad Software). A Boltzmann sigmoidal curve progression was assumed. The V50 inflexion points were calculated and the difference of the variant V50 to the wild-type measured in parallel represented the shift of the mean defolding temperature. Average reduction of the defolding temperatures in comparison to wild-type (mean defolding temperature 47.1{degree sign}C; standard error of the mean 0.25 {degree sign}C) is shown; bars indicate the standard errors of the mean.</p>