Optimized high-purity protein preparation of biologically active recombinant VacA cytotoxin variants from Helicobacter pylori.

Protein Expression and Purification
Aung Khine LinnGerd Katzenmeier

Abstract

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 proto...Continue Reading

References

Apr 1, 1991·Infection and Immunity·T L CoverM J Blaser
Jun 1, 1988·Journal of Medical Microbiology·R D LeunkD R Morgan
Aug 26, 1998·Proceedings of the National Academy of Sciences of the United States of America·C PagliacciaJ M Reyrat
Dec 23, 1999·The Journal of Biological Chemistry·A D Vinion-DubielT L Cover
Apr 7, 2000·Advances in Protein Chemistry·M Arai, K Kuwajima
Jun 26, 2002·Letters in Applied Microbiology·S A ChisholmR J Owen
Feb 4, 2003·The Journal of Biological Chemistry·Mark S McClainTimothy L Cover
Jun 9, 2004·Journal of Clinical Microbiology·Santanu ChattopadhyayAsish K Mukhopadhyay
Apr 9, 2005·The Journal of Biological Chemistry·Victor J TorresTimothy L Cover
Feb 24, 2006·Infection and Immunity·Christophe GenissetJohn L Telford
Oct 4, 2007·Proceedings of the National Academy of Sciences of the United States of America·Kelly A GangwerD Borden Lacy
Jun 10, 2010·Biochemistry·Christian González-RiveraTimothy L Cover
Jun 6, 2012·Memórias do Instituto Oswaldo Cruz·Meyssa Quezado de Figueiredo CavalcanteLucia Libanez Bessa Campelo Braga
Aug 25, 2012·Frontiers in Cellular and Infection Microbiology·Samuel L PalframanKipros Gabriel
Feb 6, 2013·Proceedings of the National Academy of Sciences of the United States of America·Mathias OertliAnne Müller
Jun 26, 2014·BioMed Research International·Muhammad JunaidGerd Katzenmeier
Apr 24, 2016·Toxicon : Official Journal of the International Society on Toxinology·Muhammad JunaidGerd Katzenmeier
Jul 7, 2016·Infection and Immunity·Christian González-RiveraTimothy L Cover
Mar 22, 2019·Proceedings of the National Academy of Sciences of the United States of America·Kaiming ZhangWah Chiu
Apr 8, 2019·Journal of Molecular Biology·Min SuMelanie D Ohi
Apr 25, 2019·Advances in Experimental Medicine and Biology·Jeannette M Whitmire, D Scott Merrell

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