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Title: Optimized high-purity protein preparation of biologically active recombinant VacA cytotoxin variants from Helicobacter pylori
Authors: Aung Khine Linn
Nitchakan Samainukul
Hui Chun Li
Chanan Angsuthanasombat
Gerd Katzenmeier
Tzu Chi University
Mahidol University
Faculty of Medicine, Siriraj Hospital, Mahidol University
Biophysics Institute for Research and Development (BIRD)
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 1-Nov-2020
Citation: Protein Expression and Purification. Vol.175, (2020)
Abstract: © 2020 Elsevier Inc. Vacuolating cytotoxin A (VacA) is a highly polymorphic virulence protein produced by the human gastric pathogen Helicobacter pylori which can cause gastritis, peptic ulcer and gastric cancer. Here, we present an optimized protein preparation of the mature full-length VacA variants (m1-and m2-types) and their 33-kDa N-terminal and 55/59-kDa C-terminal domains as biologically active recombinant proteins fused with an N-terminal His(6) tag. All recombinant VacA constructs were over-expressed in Escherichia coli as insoluble inclusions which were soluble when phosphate buffer (pH 7.4) was supplemented with 5–6 M urea. Upon immobilized-Ni2+ affinity purification under 5-M urea denaturing conditions, homogenous products (>95% purity) of 55/59-kDa domains were consistently obtained while only ~80% purity of both mature VacA variants and the 33-kDa truncate was achieved, thus requiring additional purification by size-exclusion chromatography. After successive refolding via optimized stepwise dialysis, all refolded VacA proteins were proven to possess both cytotoxic and vacuolating activity against cultured human gastric epithelial cells albeit the activity observed for VacA-m2 was lower than the m1-type variant. Such an optimized protocol described herein was effective for production of high-purity recombinant VacA proteins in large amounts (~30–40 mg per liter culture) that would pave the way for further studies on sequence-structure and function relationships of different VacA variants.
ISSN: 10960279
Appears in Collections:Scopus 2020

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