Kaltwasser Marcus, Wiegert Thomas, Schumann Wolfgang
Institute of Genetics, University of Bayreuth, D-95445 Bayreuth, Germany.
Appl Environ Microbiol. 2002 May;68(5):2624-8. doi: 10.1128/AEM.68.5.2624-2628.2002.
Here we describe the construction and application of six new tagging vectors allowing the fusion of two different types of tagging sequences, epitope and localization tags, to any Bacillus subtilis protein. These vectors are based on the backbone of pMUTIN2 and replace the lacZ gene with tagging sequences. Fusion of the tagging sequences occurs by PCR amplification of the 3' terminal part of the gene of interest (about 300 bp), insertion into the tagging vector in such a way that a fusion protein will be synthesized upon integration of the whole vector via homologous recombination with the chromosomal gene. Three of these tagging sequences (FLAG, hemagglutinin, and c-Myc) allow the covalent addition of a short epitope tag and thereby detection of the fusion proteins in immunoblots, while three other tags (green fluorescent protein(+), yellow fluorescent protein, and cyan fluorescent protein) are helpful in assigning proteins within one of the compartments of the cell. The versatility of these vectors was demonstrated by fusing these tags to the cytoplasmically located HtpG and the inner membrane protein FtsH.
在此，我们描述了六种新型标签载体的构建与应用，这些载体可使两种不同类型的标签序列（表位标签和定位标签）与任何枯草芽孢杆菌蛋白融合。这些载体基于pMUTIN2的骨架，并用标签序列取代了lacZ基因。标签序列的融合通过对感兴趣基因的3'末端部分（约300 bp）进行PCR扩增实现，以这样一种方式插入标签载体，即通过与染色体基因的同源重组将整个载体整合后会合成融合蛋白。其中三种标签序列（FLAG、血凝素和c-Myc）允许共价添加短表位标签，从而在免疫印迹中检测融合蛋白，而其他三种标签（绿色荧光蛋白（+）、黄色荧光蛋白和青色荧光蛋白）有助于在细胞的一个区室中定位蛋白质。通过将这些标签与位于细胞质中的HtpG和内膜蛋白FtsH融合，证明了这些载体的多功能性。
