Kawaguchi Nanami, Ogawa Haruko, Date Kimie
Chemistry and Biochemistry, Division of Advanced Sciences, Graduate School of Humanities and Sciences, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo, Japan.
Glycoscience Division, Institute for Human Life Innovation, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo, Japan.
Bio Protoc. 2019 Jul 5;9(13):e3286. doi: 10.21769/BioProtoc.3286.
Human pancreatic lipase (HPL) is the main lipolytic enzyme involved in the digestion of dietary fat. An active recombinant human pancreatic lipase (recHPL) was successfully prepared for the first time in an ) expression system using a short Strep-tag II (ST II). The recHPL-ST II was solubilized with 8 M urea from the lysate and purified on a Strep-Tactin-Sepharose column. After refolding by stepwise dialyses against decreasing concentrations of urea in the presence of glycerol and Ca for two days followed by gel filtration FPLC, 1.8-6 mg of active recHPL-ST II was obtained from 1 L of culture. Here we report the expression, purification, and optimized refolding procedures for active recHPL from , thus establishing it as a suitable system for the production of recHPL of high purity and scaling up.
人胰脂肪酶（HPL）是参与膳食脂肪消化的主要脂解酶。首次在使用短链抗生物素蛋白标签II（ST II）的）表达系统中成功制备了活性重组人胰脂肪酶（recHPL）。recHPL-ST II用8 M尿素从裂解物中溶解，并在链霉亲和素-琼脂糖柱上纯化。在甘油和Ca存在下，通过逐步透析到尿素浓度降低的条件下进行两天的复性，然后进行凝胶过滤快速蛋白质液相色谱（FPLC），从1 L培养物中获得了1.8 - 6 mg活性recHPL-ST II。在此我们报告了从）中表达、纯化活性recHPL的优化复性程序，从而将其确立为生产高纯度recHPL并进行放大生产的合适系统。
