Synthetic gene variants and chimeras were all made by standard in-house procedures essentially as previously described [45] (link) and cloned into a pET24a expression vector (EMD, Madison, WI) under control of the T7 promoter, between the XbaI and EcoRI restriction sites. Each construct was completely sequenced in both directions to ensure consistency with the designed sequence. Each variant plasmid was transformed into E. coli expression host strain BL21(DE3) pLysS (Invitrogen, Carlsbad, CA). BL21(DE3) pLysS was chosen as the host for all expression studies described. The low-level expression in this host of T7 lysozyme, an inhibitor of T7 RNA polymerase, gives tight repression of heterologous expression prior to induction to minimize potential gene toxicity which could affect data quality.
Prior to protein expression analysis of the variants, expression was analyzed for multiple variants showing a range of expression levels to determine appropriate expression time and temperature. Strong, consistent expression was achieved at 30°C, a commonly used temperature for heterologous expression in E. coli. Time courses at 30°C showed expressed protein levels increasing to a maximum after approximately two hours, as the cells entered stationary phase growth, and expression remained steady for at least five hours. Relative protein expression levels between these variants were consistent as protein accumulated during growth phase and were maintained in stationary phase (data not shown). For our variant analysis we chose to express for four hours at 30°C.
At least three independent isolates for each gene were picked and cultured overnight in 2 ml Luria Broth (LB) containing 25 µg/ml kanamycin and 25 µg/ml chloramphenicol. Overnight cultures were diluted 50-fold in fresh media and incubated at 37°C until the cells were in mid-log growth (OD at 600 nm = 0.6). Expression was induced by addition of IPTG to 1 mM and incubation for four hours at 30°C. Final optical densities of cultures were measured and equivalent amounts of culture were analyzed by polyacrylamide gel electrophoresis. Gels were stained with Sypro Ruby (Pierce), visualized by fluorescence imaging, and protein band intensities quantified using TotalLab100 image analysis software (Nonlinear, Inc). Each gel contained protein concentration standards to calibrate band intensity. In each experiment, a consistent reference variant was co-expressed in triplicate. For analysis of polymerase expression, the reference was a phi29 DNA polymerase variant identical to variant 21 but containing two differences in the 5′ untranslated region. For analysis of the scFv variants, Variant A13 was used as the reference. Reference variants were used to correct for experiment to experiment variation in yield. Measured expression levels are all relative to these references. Reported values in µg/ml are normalized to the average expression level of the references over the sum of experiments. The detection limit of the assay was approximately 5 µg protein per ml culture at an A600 = 3. The standard error of measured expression for variant repeats was generally <20% of the mean.
Prior to protein expression analysis of the variants, expression was analyzed for multiple variants showing a range of expression levels to determine appropriate expression time and temperature. Strong, consistent expression was achieved at 30°C, a commonly used temperature for heterologous expression in E. coli. Time courses at 30°C showed expressed protein levels increasing to a maximum after approximately two hours, as the cells entered stationary phase growth, and expression remained steady for at least five hours. Relative protein expression levels between these variants were consistent as protein accumulated during growth phase and were maintained in stationary phase (data not shown). For our variant analysis we chose to express for four hours at 30°C.
At least three independent isolates for each gene were picked and cultured overnight in 2 ml Luria Broth (LB) containing 25 µg/ml kanamycin and 25 µg/ml chloramphenicol. Overnight cultures were diluted 50-fold in fresh media and incubated at 37°C until the cells were in mid-log growth (OD at 600 nm = 0.6). Expression was induced by addition of IPTG to 1 mM and incubation for four hours at 30°C. Final optical densities of cultures were measured and equivalent amounts of culture were analyzed by polyacrylamide gel electrophoresis. Gels were stained with Sypro Ruby (Pierce), visualized by fluorescence imaging, and protein band intensities quantified using TotalLab100 image analysis software (Nonlinear, Inc). Each gel contained protein concentration standards to calibrate band intensity. In each experiment, a consistent reference variant was co-expressed in triplicate. For analysis of polymerase expression, the reference was a phi29 DNA polymerase variant identical to variant 21 but containing two differences in the 5′ untranslated region. For analysis of the scFv variants, Variant A13 was used as the reference. Reference variants were used to correct for experiment to experiment variation in yield. Measured expression levels are all relative to these references. Reported values in µg/ml are normalized to the average expression level of the references over the sum of experiments. The detection limit of the assay was approximately 5 µg protein per ml culture at an A600 = 3. The standard error of measured expression for variant repeats was generally <20% of the mean.
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