Expression of the 14kDa protein azurin with different tags and promoters
The bacterial 14 kDa-protein “azurin” was cloned and expressed by means of 21 different E. coli specific StarGate® Acceptor Vectors. Comparable amounts of E. coli cells were harvested 3 hours after induction of protein expression with anhydrotetracycline in case of pASG vectors containing the tightly controlled tet-promoter and with IPTG in case of pPSG vectors, containing the T7-promoter. Then, cell samples were lysed with gel loading buffer, heated for 5 min to 95 °C and then subjected to 15 % SDS-PAGE with subsequent Coomassie staining. Periplasmic secretion by means of ompA led in all cases to accumulation of comparable amounts of the protein of interest (lanes 11-15). This could be expected as azurin is also secreted in its authentic host P. aeruginosa. In case of cytosolic expression, however, interesting aspects became obvious since it was not expected that cytosolic expression of azurin is possible at all. Expression was enabled in case of N-terminal truncation by fusion of an affinity tag and tightly controlled expression of the tet promoter (lanes 2,3,7,8 and 9). This may be e.g. explained by some unusual secondary structure of the mRNA when the sequence encoding the mature protein is brought into close proximity to the 5’ untranslated region containing the Shine/Dalgarno sequence which leads to poor initiation of translation. Further, in case of small tags having only low influence of azurin folding/degradation, the use of the tightly regulated tet-promoter seems to be clearly preferable over the strong T7 promoter (compare lanes 2 and 3 with lanes 17 and 18 where the same protein construct is expressed with different success). Leaky expression of azurin in case of pPSG vectors may exert some toxic effect leading to counter selection and plasmid loss prior to induction of expression. In case of fusion of GST, however, the strong T7 promoter provides a higher level of expression (compare lane 4 with lane 19). This may be explained by the presence of the larger fusion partner GST. GST is in contrast to a peptide tag an efficiently folding, stable protein which obviously overcomes the adverse characteristics of the smaller protein “azurin” so that the stronger T7 promoter becomes effective in this case - in spite of poor regulation. With this comparison, the authors want in no way favour or downgrade any of the shown expression and/or purification systems as the situation may be completely different for another protein of interest (data not shown). It is only to show that initial screening for expression conditions may be worthwhile and may lead to unexpected results.
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