Kerschbaumer R J, Hirschl S, Schwager C, Ibl M, Himmler G
Institute of Applied Microbiology, University of Agriculture and Forestry, Vienna, Austria.
Immunotechnology. 1996 Jun;2(2):145-50. doi: 10.1016/1380-2933(96)00040-1.
Expression of enzymatically active protein fusions in Escherichia coli could facilitate the analysis of proteins and even replace some reagents frequently used in immunology such as chemically produced antibody-enzyme conjugates. For this purpose there is up to now no system of general utility available.
The vector pDAP2 has been designed for simplified fusion of single-chain Fv fragments to the N-terminus of E. coli alkaline phosphatase. The resulting immunoconjugates can be produced by expression in E. coli and purified in a single step via metal affinity chromatography.
Several different single-chain Fv genes as well as peptide coding oligonucleotides have been cloned into pDAP2 and tested for expression levels, purification properties and usefulness in ELISA and immunowestern blotting.
The fusion proteins from pDAP2 can be prepared at levels of several milligrams per liter culture from the periplasma of the cells. The proteins work well in different immunoassay formats such as ELISA or western blotting.
pDAP2 is compatible to phage display vectors such as pHEN1, pCOCK and the pCANTAB-series (Pharmacia, Sweden). Genes of single-chain antibody fragments can be simply swapped from these vectors into pDAP2. Furthermore, we demonstrate that it is possible to produce alkaline phosphatase-active peptides with this vector by inserting synthetic coding oligonucleotides. This allows simple analysis of the binding properties of peptides and may have some advantages over other systems such as fusion to glutathione-S-transferase.
在大肠杆菌中表达具有酶活性的蛋白质融合体有助于蛋白质分析，甚至可替代免疫学中常用的一些试剂，如化学合成的抗体 - 酶偶联物。但目前尚无通用的系统可实现此目的。
设计载体pDAP2，用于将单链Fv片段简化融合至大肠杆菌碱性磷酸酶的N端。通过在大肠杆菌中表达，所得免疫偶联物可通过金属亲和层析一步纯化。
将几种不同的单链Fv基因以及编码肽的寡核苷酸克隆到pDAP2中，并检测其表达水平、纯化特性以及在ELISA和免疫印迹中的实用性。
来自pDAP2的融合蛋白可从细胞周质中以每升培养物数毫克的水平制备。这些蛋白在不同的免疫分析形式如ELISA或western印迹中表现良好。
pDAP2与噬菌体展示载体如pHEN1、pCOCK和pCANTAB系列（瑞典Pharmacia公司）兼容。单链抗体片段基因可简单地从这些载体交换到pDAP2中。此外，我们证明通过插入合成编码寡核苷酸，用该载体可产生具有碱性磷酸酶活性的肽。这允许对肽的结合特性进行简单分析，并且可能比其他系统如与谷胱甘肽 - S -转移酶融合具有一些优势。
