Department of Biochemistry, University of Bayreuth, Universitätsstr. 30, 95447, Bayreuth, Germany.
Extremophiles. 2010 May;14(3):249-59. doi: 10.1007/s00792-010-0304-9. Epub 2010 Mar 12.
Central to genetic work in any organism are the availability of a range of inducible and constitutive promoters. In this work we studied several promoters for use in the hyperthermophilic archaeon Sulfolobus acidocaldarius. The promoters were tested with the aid of an E. coli-Sulfolobus shuttle vector in reporter gene experiments. As the most suitable inducible promoter a maltose inducible promoter was identified. It comprises 266 bp of the sequence upstream of the gene coding for the maltose/maltotriose binding protein (mbp, Saci_1165). Induction is feasible with either maltose or dextrin at concentrations of 0.2-0.4%. The highest increase in expression (up to 17-fold) was observed in late exponential and stationary phase around 30-50 h after addition of dextrin. Whereas in the presence of glucose and xylose higher basal activity and reduced inducibility with maltose is observed, sucrose can be used in the growth medium additionally without affecting the basal activity or the inducibility. The minimal promoter region necessary could be narrowed down to 169 bp of the upstream sequence. The ABCE1 protein from S. solfataricus was successfully expressed under control of the inducible promoter with the shuttle vector pC and purified from the S. acidocaldarius culture with a yield of about 1 mg L(-1) culture. In addition we also determined the promoter strength of several constitutive promoters.
在任何生物体的遗传工作中，核心问题是一系列可诱导和组成型启动子的可用性。在这项工作中，我们研究了几种启动子，用于嗜热古菌 Sulfolobus acidocaldarius。使用大肠杆菌- Sulfolobus 穿梭载体在报告基因实验中测试了这些启动子。作为最适合的诱导启动子，确定了麦芽糖诱导启动子。它由编码麦芽糖/麦芽三糖结合蛋白（mbp，Saci_1165）的基因上游的 266 bp 序列组成。用麦芽糖或糊精在 0.2-0.4%的浓度下都可以进行诱导。在添加糊精后 30-50 小时左右的指数晚期和静止期观察到表达的最高增加（高达 17 倍）。而在存在葡萄糖和木糖的情况下，观察到麦芽糖的基础活性较高和诱导能力降低，而蔗糖可以在生长培养基中另外使用而不影响基础活性或诱导能力。必需的最小启动子区域可以缩小到上游序列的 169 bp。来自 S. solfataricus 的 ABCE1 蛋白在穿梭载体 pC 的诱导启动子控制下成功表达，并从 S. acidocaldarius 培养物中纯化，产量约为 1 mg L(-1) 培养物。此外，我们还确定了几个组成型启动子的启动子强度。
