Nakajima Daisuke, Saito Kenji, Yamakawa Hisashi, Kikuno Reiko F, Nakayama Manabu, Ohara Reiko, Okazaki Noriko, Koga Hisashi, Nagase Takahiro, Ohara Osamu
Department of Human Gene Research, Kazusa DNA Research Institute, Kazusa-Kamatari, Kisarazu, Chiba, Japan.
DNA Res. 2005;12(4):257-67. doi: 10.1093/dnares/dsi010.
Although we have so far identified and sequenced >2000 human long cDNAs, known as KIAA cDNAs, half of them have yet to be functionally annotated. Expression-ready cDNA clones derived from these genes, where the open reading frame (ORF) of the gene of interest is placed under the control of an appropriate promoter, are critical for functional characterization of these gene products. In this study, we attempted to systematically convert original cDNA clones to expression-ready forms for native and fusion proteins. For this purpose, we developed a new method for ORF cloning based on a homologous recombination in Escherichia coli to avoid laborious manipulations and artificial introduction of mutations in ORF. Using 1589 putative full-length ORFs (from 1002 KIAA genes, 119 human known genes and 468 mouse genes) with an average size of 2.8 kb, we successfully prepared expression plasmids for 1463 native proteins and for 1343 fusion proteins by this method. The resultant expression-ready clones were examined using an in vitro transcription/translation system followed by SDS-polyacrylamide gel electrophoresis and by transient expression of GFP-fusion proteins in human embryonic kidney (HEK) 293 cells. This set of expression-ready clones of long cDNAs encoding large proteins would open a new route to experimentally analyze their functions on a proteomic scale, since unavailability of expression-ready clones for mammalian large proteins has been a major obstacle to the functional analysis of these cDNAs.
尽管到目前为止我们已经鉴定并测序了2000多个已知为KIAA cDNA的人类长链cDNA，但其中一半尚未进行功能注释。从这些基因衍生而来的可供表达的cDNA克隆，其中目的基因的开放阅读框（ORF）置于合适的启动子控制之下，对于这些基因产物的功能表征至关重要。在本研究中，我们试图系统地将原始cDNA克隆转化为可供天然蛋白和融合蛋白表达的形式。为此，我们基于大肠杆菌中的同源重组开发了一种新的ORF克隆方法，以避免繁琐的操作和ORF中人工引入突变。利用1589个推定的全长ORF（来自1002个KIAA基因、119个人类已知基因和468个小鼠基因），平均大小为2.8 kb，我们通过该方法成功制备了1463种天然蛋白和1343种融合蛋白的表达质粒。使用体外转录/翻译系统，随后进行SDS-聚丙烯酰胺凝胶电泳以及在人胚肾（HEK）293细胞中瞬时表达GFP融合蛋白，对所得的可供表达的克隆进行了检测。这组编码大蛋白的长链cDNA的可供表达的克隆将为在蛋白质组学规模上实验分析它们的功能开辟一条新途径，因为缺乏可供表达的克隆一直是这些cDNA功能分析的主要障碍。
