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A novel protocol for the preparation of active recombinant human pancreatic lipase from Escherichia coli.

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.

SUBMITTER: Kawaguchi N

PROVIDER: S-EPMC6267337 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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A novel protocol for the preparation of active recombinant human pancreatic lipase from Escherichia coli.

Kawaguchi Nanami N Date Kimie K Suzuki Yusuke Y Tomita Chihiro C Naradate Rina R Higami Tomoko T Nakamura Kosuke K Aikawa Kyoko K Ogawa Haruko H

Journal of biochemistry 20181201 6

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-glycosyl ...[more]

PMID: 30101295

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Project description:BackgroundRecombinant protein-based therapeutics have become indispensable for the treatment of many diseases. They are produced using well-established expression systems based on bacteria, yeast, insect and mammalian cells. The majority of therapeutic proteins are glycoproteins and therefore the post-translational attachment of sugar residues is required. The development of an engineered Escherichia coli-based expression system for production of human glycoproteins could potentially lead to increased yields, as well as significant decreases in processing time and costs.ResultsThis work describes the expression of functional human-derived glycosyltransferase UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 2 (GalNAcT2) in a recombinant E. coli strain. For expression, a codon-optimised gene encoding amino acids 52-571 of GalNAcT2 lacking the transmembrane N-terminal domain was inserted into a pET-23 derived vector encoding a polyhistidine-tag which was translationally fused to the N-terminus of the glycosyltransferase (HisDapGalNAcT2). The glycosyltransferase was produced in E. coli using a recently published expression system. Soluble HisDapGalNAcT2 produced in SHuffle® T7 host cells was purified using nickel affinity chromatography and was subsequently analysed by size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) and circular dichroism spectroscopy to determine molecular mass, folding state and thermal transitions of the protein. The activity of purified HisDapGalNAcT2 was monitored using a colorimetric assay based on the release of phosphate during transfer of glycosyl residues to a model acceptor peptide or, alternatively, to the granulocyte-colony stimulating growth factor (G-CSF). Modifications were assessed by Matrix Assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry analysis (MALDI-TOF-MS) and Electrospray Mass Spectrometry analysis (ESI-MS). The results clearly indicate the glycosylation of the acceptor peptide and of G-CSF.ConclusionIn the present work, we isolated a human-derived glycosyltransferase by expressing soluble HisDapGalNAcT2 in E. coli. The functional activity of the enzyme was shown in vitro. Further investigations are needed to assess the potential of in vivo glycosylation in E. coli.

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A novel protocol for the preparation of active recombinant human pancreatic lipase from Escherichia coli.

Publications

A novel protocol for the preparation of active recombinant human pancreatic lipase from Escherichia coli.

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