期刊
BIO-PROTOCOL
卷 9, 期 13, 页码 -出版社
BIO-PROTOCOL
DOI: 10.21769/BioProtoc.3286
关键词
Human pancreatic lipase; Escherichia coli; Lipolytic activity; Refolding; Strep-tag II; Gel filtration
类别
资金
- Daiwa Securities Health Foundation
- Japan Foundation of Applied Enzymology
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 Escherichia coli (E. cob) expression system using a short Strep-tag II (ST II). The recHPL-ST II was solubilized with 8 M urea from the E. coli 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 Ca2+ 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 E. coli, thus establishing it as a suitable system for the production of recHPL of high purity and scaling up.
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