Vasina J A, Baneyx F
Department of Chemical Engineering, University of Washington, Seattle 98195, USA.
Protein Expr Purif. 1997 Mar;9(2):211-8. doi: 10.1006/prep.1996.0678.
The aggregation-prone fusion protein preS2-S'-beta-galactosidase was used as a model system to compare the efficiencies of the IPTG-inducible tac promoter and the low-temperature-inducible cspA promoter in directing the expression of soluble recombinant polypeptides at reduced growth temperatures in Escherichia coli. At 37 degrees C, the fusion protein was produced at high levels from the tac promoter, but aggregated quantitatively in a biologically inactive form. In contrast, little preS2-S'-beta-galactosidase was synthesized from the cspA promoter at this temperature, presumably due to transcript instability. The highest yields of active enzyme were obtained following temperature downshift from 37 to 30 degrees C for the tac promoter and 25 degrees C for the cspA promoter. At 25 degrees C, the kinetics of accumulation of beta-galactosidase activity, ratios of soluble to insoluble fusion protein, and synthesis rates of preS2-S'-beta-galactosidase were virtually identical for both promoters for a period of 2 h postinduction. Thereafter, the cspA promoter became repressed, whereas synthesis of the fusion protein continued with the tac system. Following transfer to 10 degrees C, the tac promoter was almost completely inhibited while the cspA promoter was able to direct the synthesis of soluble preS2-S'-beta-galactosidase for up to 2 h. However, the levels of active enzyme produced were approximately threefold lower than those measured at 25 degrees C. Overexpression of native CspA had no effect on the accumulation of active preS2-S'-beta-galactosidase from the cspA promoter. It is therefore unlikely that CspA acts as it own positive inducer. Our results indicate that the cspA promoter can efficiently substitute for the tac system at 25 degrees C and may be particularly valuable for the expression of highly aggregation-prone or unstable gene products at 10 degrees C.
以易聚集的融合蛋白前S2-S'-β-半乳糖苷酶作为模型系统，比较异丙基-β-D-硫代半乳糖苷（IPTG）诱导型tac启动子和低温诱导型cspA启动子在大肠杆菌中降低生长温度时指导可溶性重组多肽表达的效率。在37℃时，融合蛋白由tac启动子高水平产生，但以生物学无活性的形式定量聚集。相反，在此温度下，cspA启动子合成的前S2-S'-β-半乳糖苷酶很少，推测是由于转录本不稳定。对于tac启动子，从37℃降至30℃，对于cspA启动子，从37℃降至25℃后，获得了最高产量的活性酶。在25℃时，诱导后2小时内，两种启动子的β-半乳糖苷酶活性积累动力学、可溶性与不溶性融合蛋白的比例以及前S2-S'-β-半乳糖苷酶的合成速率几乎相同。此后，cspA启动子被抑制，而融合蛋白的合成在tac系统中继续。转移到10℃后，tac启动子几乎完全被抑制，而cspA启动子能够指导可溶性前S2-S'-β-半乳糖苷酶的合成长达2小时。然而，产生的活性酶水平比在25℃时测得的低约三倍。天然CspA的过表达对cspA启动子活性前S2-S'-β-半乳糖苷酶的积累没有影响。因此，CspA不太可能作为其自身的正诱导剂。我们的结果表明，cspA启动子在25℃时可以有效地替代tac系统，对于在10℃时表达高度易聚集或不稳定的基因产物可能特别有价值。
