A novel protocol for the preparation of active recombinant human pancreatic lipase from Escherichia coli. (8th August 2018)

• Record Type: Journal Article
• Authors: Kawaguchi, Nanami; Date, Kimie; Suzuki, Yusuke; Tomita, Chihiro; Naradate, Rina; Higami, Tomoko; Nakamura, Kosuke; Aikawa, Kyoko; Ogawa, Haruko
• Is Part Of: Journal of biochemistry. Volume 164:Number 6(2018:Dec.)
• Page Start: 407
• Page End: 414
• Publication Date: 2018-08-08
• Subjects: Escherichia coli -- human pancreatic lipase -- lipolytic activity -- refolding -- Strep-tag II; Biochemistry -- Periodicals; Biochemistry -- Periodicals; Electronic journals; 572.05
• DOI: 10.1093/jb/mvy067
• Languages: English
• ISSNs: 0021-924X

Abstract

Abstract: An active recombinant human pancreatic lipase (recHPL) was successfully prepared for the first time from the Escherichia coli expression system using short Strep-tag II (ST II). The recHPL–ST II was solubilized using 8 M urea from E.coli lysate and purified on a Strep-Tactin-Sepharose column. After refolding by stepwise dialyses in the presence of glycerol and Ca 2+ for 2 days followed by gel filtration, 1.8–6 mg of active recHPL–ST II was obtained from 1 L of culture. The recHPL was non-glycosylated, but showed almost equal specific activity, pH-dependency and time-dependent stability compared to those of native porcine pancreatic lipase (PPL) at 37°C. However, the recHPL lost its lipolytic activity above 50°C, showing a lower heat-stability than that of native PPL, which retained half its activity at this temperature.