Publication: Optimized high-purity protein preparation of biologically active recombinant VacA cytotoxin variants from Helicobacter pylori
Issued Date
2020-11-01
Resource Type
ISSN
10960279
10465928
10465928
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2-s2.0-85088215140
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Mahidol University
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SCOPUS
Bibliographic Citation
Protein Expression and Purification. Vol.175, (2020)
Suggested Citation
Aung Khine Linn, Nitchakan Samainukul, Hui Chun Li, Chanan Angsuthanasombat, Gerd Katzenmeier Optimized high-purity protein preparation of biologically active recombinant VacA cytotoxin variants from Helicobacter pylori. Protein Expression and Purification. Vol.175, (2020). doi:10.1016/j.pep.2020.105696 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/57670
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Title
Optimized high-purity protein preparation of biologically active recombinant VacA cytotoxin variants from Helicobacter pylori
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.
