Ganesan Mala, Paithankar Khanderao R, Jagannadham Medicharla V, Sundaram Curam S, Murthy Bulusu S, Singh Lalji
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.
Protein Expr Purif. 2007 May;53(1):164-78. doi: 10.1016/j.pep.2006.11.007. Epub 2006 Nov 18.
DNA-binding proteins play pivotal roles in transcription, DNA-replication, recombination/repair and determine cell-fate in all physiological conditions of differentiation, development and disease. As they are present in extremely small amounts in cells, their isolation/identification, particularly from scarce tissues is impracticable. We cloned the cDNA pool of snake (Ptyas mucosus) oocytes (a scarce tissue) in bacteria, overexpressed total library, purified and identified DNA-binding proteins expressed in the library. Although snake databases do not exist, we identified 23 DNA-binding proteins, obtained 10-15 amino acids internal sequence tags of six of them and succeeded in PCR amplification of the cDNAs of five proteins. We employed electro spray ionization mass spectrometry, matrix assisted laser desorption/ionization time of flight and analyzed the results by peptide mass fingerprint (PMF) and various sequence BLAST analyses. Proteins identified were largely unanimous between the PMF and BLAST analyses. We expect these proteins to play important roles in snake embryonic development and differentiation. We arrived at homologous mouse proteins to some of the identified snake proteins and are working towards characterizing their structure and physiological function. Similar approaches shall prove valuable in isolation and identification of important factors from scarce carcinoma tissues, mammalian oocytes and early embryos, which might be involved in important functions like nuclear reprogramming, embryonic development and differentiation.
DNA结合蛋白在转录、DNA复制、重组/修复过程中发挥着关键作用，并在分化、发育和疾病的所有生理条件下决定细胞命运。由于它们在细胞中的含量极少，因此将其分离/鉴定出来，尤其是从稀少的组织中分离/鉴定出来，是不切实际的。我们将蛇（滑鼠蛇）卵母细胞（一种稀少的组织）的cDNA文库克隆到细菌中，对整个文库进行过表达，纯化并鉴定文库中表达的DNA结合蛋白。尽管不存在蛇的数据库，但我们鉴定出了23种DNA结合蛋白，获得了其中6种蛋白的10 - 15个氨基酸的内部序列标签，并成功对5种蛋白的cDNA进行了PCR扩增。我们采用了电喷雾电离质谱、基质辅助激光解吸/电离飞行时间质谱，并通过肽质量指纹图谱（PMF）和各种序列比对分析对结果进行分析。在PMF和比对分析之间，鉴定出的蛋白质在很大程度上是一致的。我们预计这些蛋白在蛇胚胎发育和分化中发挥重要作用。我们已找到一些已鉴定出的蛇蛋白的同源小鼠蛋白，并正在努力表征它们的结构和生理功能。类似的方法在从稀少的癌组织、哺乳动物卵母细胞和早期胚胎中分离和鉴定可能参与核重编程、胚胎发育和分化等重要功能的重要因子方面将被证明是有价值的。
