| 摘要 |
PURPOSE: An expression vector including human tissue factor (HTF) gene and a method for manufacturing HTF fragment using it are provided. HTF can be expressed and purified in the simple and economical method by the vector including the minimal fragment of HTF with the coagulating activity. CONSTITUTION: An expression vector includes the sequence which corresponds to the extracellular domain (1-219 amino acids) of human tissue factor. This extracellular domain has the same coagulating activity as the intact human tissue factor. The complicated chromatography process for the maintenance of enzyme activity and the cleavage process of HTF are unnecessary because HTF fragment is purified in the form of the inclusion body. HTF fragment is purified by the following steps: 1) the cells are cultured until A595 is 0.5 and induced by IPTG, 2) the cultured cells are frozen, 3) the frozen cells are lysed by the sonication, 4) the inclusion bodies are purified, 5) the insoluble components are removed by the centrifugation after the inclusion bodies are dissolved in buffer, 6) the dialysis is performed.
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