Prein B, Natter K, Kohlwein S D
SFB Biomembrane Research Center, Department of Biochemistry, Technical University Graz, Petersgasse 12/2, A-8010 Graz, Austria.
FEBS Lett. 2000 Nov 17;485(1):29-34. doi: 10.1016/s0014-5793(00)02179-7.
A novel rapid polymerase chain reaction (PCR)-based technique for N-terminal attachment of green fluorescent protein (GFP) to a yeast protein is described. Genomic integration of a PCR-generated loxPkanMX4loxP-yEGFP fusion cassette immediately upstream of the open reading frame (ORF) allows for selection of G418 resistant transformants carrying GFP fused N-terminally to the protein of interest. In a subsequent step, the loxPkanMX4loxP selection marker that is inserted between the tagged ORF and the endogenous promoter is excised upon site-specific recombination between the loxP sites by Cre recombinase, leaving behind in the promoter one loxP site, immediately upstream of the GFP start codon. The essential protein Ydl193wp of unknown function and the oleate-inducible fatty acid activation protein, encoded by FAA2, were N-terminally tagged using the novel technique. Both experiments yielded viable haploid strains with growth phenotypes indistinguishable from the wild type strain. The subcellular localization pattern for the chromosomally expressed GFP-Ydl193wp to the endoplasmic reticulum and lipid particles was identical to the pattern observed for a plasmid-borne GFP construct expressed under control of the MET25(p) promoter, albeit at a lower level and with a more homogeneous distribution among the cell population. GFP-FAA2 was inducible by oleate, as is the wild type gene, demonstrating that specific expression patterns are not grossly affected by the promoter manipulation. In agreement with previous reports, GFP-Faa2p was found to localize to peroxisomes.
本文描述了一种基于聚合酶链反应（PCR）的新技术，用于将绿色荧光蛋白（GFP）N端连接到酵母蛋白上。通过PCR生成的loxPkanMX4loxP-yEGFP融合盒在开放阅读框（ORF）上游进行基因组整合，可筛选出携带N端与目标蛋白融合的GFP的G418抗性转化体。在后续步骤中，通过Cre重组酶在loxP位点之间进行位点特异性重组，切除插入在标记的ORF和内源启动子之间的loxPkanMX4loxP选择标记，在启动子中留下一个loxP位点，紧挨着GFP起始密码子的上游。使用该新技术对功能未知的必需蛋白Ydl193wp和由FAA2编码的油酸诱导型脂肪酸激活蛋白进行了N端标记。两个实验均产生了生长表型与野生型菌株无明显差异的有活力的单倍体菌株。染色体表达的GFP-Ydl193wp在内质网和脂质颗粒中的亚细胞定位模式与在MET25(p)启动子控制下表达的质粒携带的GFP构建体所观察到的模式相同，尽管表达水平较低且在细胞群体中的分布更均匀。GFP-FAA2如野生型基因一样可被油酸诱导，表明特定的表达模式不会受到启动子操作的严重影响。与先前的报道一致，发现GFP-Faa2p定位于过氧化物酶体。
