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Accurate Disulfide Formation in Escherichia coli : Overexpression and Characterization of the First Domain (HF6478) of the Multiple Kazal-Type Inhibitor LEKTI

Title data

Lauber, Thomas ; Marx, Ute C. ; Schulz, Axel ; Kreutzmann, Peter ; Rösch, Paul ; Hoffmann, Silke:
Accurate Disulfide Formation in Escherichia coli : Overexpression and Characterization of the First Domain (HF6478) of the Multiple Kazal-Type Inhibitor LEKTI.
In: Protein Expression and Purification. Vol. 22 (June 2001) Issue 1 . - pp. 108-112.
ISSN 1046-5928
DOI: https://doi.org/10.1006/prep.2001.1415

Abstract in another language

The human hemofiltrate peptide HF6478, a putative serine proteinase inhibitor, which is part of the precursor protein LEKTI, was cloned, overexpressed, and purified. HF6478 contains two disulfide bridges with 1-4, 2-3 connectivity, sharing partial homology to Kazal-type domains and other serine proteinase inhibitors. It was expressed as thioredoxin (Trx) fusion protein, and disulfide formation occurred in the oxidative cytoplasm of Escherichia coli Origami (DE3) strain which carries a trxB(-)/gor522(-) double mutation. The soluble fusion protein was purified using metal-chelating affinity chromatography. Cleavage of the Trx fusion protein with factor Xa and subsequent purification yielded the final product in amounts sufficient for structural studies. Characterization of recombinant HF6478 was done by amino acid sequencing, mass spectrometry, capillary zone electrophoresis, and CD spectroscopy. Taking the blood filtrate peptide HF6478 as example, we present a strategy which should facilitate the expression of different extracellular proteins in the E. coli cytoplasm.

Further data

Item Type: Article in a journal
Refereed: Yes
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors > Chair Biopolymers - Univ.-Prof. Dr. Paul Rösch
Faculties
Faculties > Faculty of Biology, Chemistry and Earth Sciences
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biopolymers
Result of work at the UBT: Yes
DDC Subjects: 500 Science > 540 Chemistry
500 Science > 570 Life sciences, biology
Date Deposited: 25 Jan 2019 09:05
Last Modified: 16 May 2019 05:37
URI: https://eref.uni-bayreuth.de/id/eprint/47028