Figure 2

Expression and solubility of GST-TSAd SH2 domains are influenced by flanking sequence and growth temperature. A. Schematic overview of the GST-fusion SH2 constructs (given in Table 1) used in this study. B. GST-fusion SH2 constructs of TSAd (1-TD, TSAd-67-207-IVTD) and Lck (Lck-SH2, Lck-124-228-RPHRD) were expressed in E.coli at room temperature (RT). Equal volumes of resuspended pellet (P), soluble fraction (S) and glutathione beads (B) were separated by 10% SDS-PAGE. Proteins were visualised by Coomassie Brilliant Blue staining. C. Quantitation of the amount of soluble GST-SH2 proteins shown in B, by immunoblotting with anti-GST and comparison to defined amounts of GST. 5 μl of 2 ml TSAd-SH2 soluble fraction and 0,06 μl of 2 ml Lck-SH2 soluble fraction from 100 ml bacterial cultures were applied on the gel. GST = 1 represents a total amount of 0,11 μg GST applied on the gel. D. Quantitation of soluble GST-SH2 proteins using Image J analysis based on C. E and F. Yield of expression of the six GST-TSAd SH2 domain constructs in E.coli at RT or 15°C, respectively. Gels processed as in B. Constructs are indicated by their short names as listed in Table 1.