COMPARISON OF SCHIZOSACCHAROMYCES-POMBE EXPRESSION SYSTEMS
COMPARISON OF SCHIZOSACCHAROMYCES-POMBE EXPRESSION SYSTEMS
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DOI：
10.1093/nar/21.12.2955
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发表时间：
1993-06-25
影响因子：
14.9
通讯作者：
FORSBURG, SL
中科院分区：
文献类型：
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作者：
FORSBURG, SL
酵母中各种问题的分析依赖于在异源启动子控制下的目的蛋白质的异位表达。在裂殖酵母S. pombe中，表达质粒相对较少，并且大多数关于其活性的报道都没有可比性。因此，难以确定使用不同表达载体进行的实验的可比性。当表征新的启动子时，同样难以确定它们相对于先前鉴定的那些启动子的活性。为了更好地评估相对启动子强度，我比较了几个诱导型和组成型表达系统在裂殖酵母中的活性，通过使用β-半乳糖苷酶报告基因。这使得一个更明智的选择适当的表达系统的基因功能分析在S。比较了四种可调控启动子和两种组成型启动子。三种受调节的启动子衍生自强大的nmtl启动子，其首先由Maundrell（1）描述，随后由Basi et al.（二）、另一个调节系统由Faryar和Gatz描述的四环素诱导系统构建（3）。两个组成型启动子是先前描述的载体pART 1（含有adh启动子; (4)和pSM 1（含有SV 40启动子; 5）。含有硫胺素诱导型nmtl启动子（1）的表达载体REP 3及其衍生物REP 41和REP 81（2）由于突变而具有较低水平的活性，它们都在其多接头内含有ATG。通过插入Xho连接体破坏了这一点;这些衍生物被称为REP 3X（全强度nmtl）、REP 41 X（稍弱; nmtl*）和REP 81 X（更弱; nmtl *）。
Analysis of a variety of problems in yeast relies on ectopic expression of the protein of interest under control of a heterologous promoter. In the fission yeast S. pombe, there are relatively few expression plasmids and most reports of their activity have not been comparative. It is thus difficult to determine the comparability of experiments carried out using different expression vectors. When new promoters are characterised, it is likewise difficult to determine their activity relative to those previously identified. In order to better assess relative promoter strengths, I have compared the activity of several inducible and constitutive expression systems in fission yeast by using a betagalactosidase reporter gene. This allows a more informed choice of appropriate expression systems for analysis of gene function in S. pombe.Four regulatable and two constitutive promoters were compared. Three regulated promoters were derived from the powerful nmtl promoter, first described by Maundrell (1) and subsequently attenuated with mutations in the TATA box by Basi et al.(2). The other regulated system was constructed from the tetracycline-inducible system described by Faryar and Gatz (3). The two constitutive promoters were the previously described vectors pART1 (containing the adh promoter;.(4) and pSMl (containing the SV40 promoter; 5). The expression vector REP3, containing the thiamine-inducible nmtl promoter (1), and its derivatives REP41 and REP81 (2), which have lower levels of activity due to mutation, all contain an ATG within their polylinker. This was destroyed by insertion ofa Xho linker; these derivatives are called REP3X (full strength nmtl), REP41X (slightly weaker; nmtl*) and REP81X (much