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 Ca2+ 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.
MeSH terms
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Animals
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Circular Dichroism
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Dietary Supplements / adverse effects
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Enzyme Inhibitors / pharmacology
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Enzyme Replacement Therapy / adverse effects
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Enzyme Stability
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Escherichia coli / growth & development
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Escherichia coli / metabolism
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Glycosylation
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Hot Temperature / adverse effects
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Humans
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Inclusion Bodies / enzymology
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Inclusion Bodies / metabolism
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Kinetics
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Lipase / adverse effects
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Lipase / antagonists & inhibitors
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Lipase / chemistry
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Lipase / genetics
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Lipase / isolation & purification
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Lipase / metabolism*
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Oligopeptides / chemistry
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Oligopeptides / genetics
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Oligopeptides / isolation & purification
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Oligopeptides / metabolism
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Orlistat / pharmacology
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Protein Conformation
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Protein Processing, Post-Translational
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Protein Refolding
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / isolation & purification
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Recombinant Fusion Proteins / metabolism*
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Solubility
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Sus scrofa
Substances
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Enzyme Inhibitors
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Oligopeptides
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Recombinant Fusion Proteins
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Trp-Ser- His-Pro-Gln-Phe-Glu-Lys
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Orlistat
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Lipase
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PNLIP protein, human