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.

空泡细胞毒素A（VacA）是一种由人胃病原体幽门螺杆菌产生的高度多态性的毒性蛋白，可引起胃炎，消化性溃疡和胃癌。在这里，我们提出了一种成熟的全长VacA变体（m1和m2型）及其33-kDa N-末端和55 / 59-kDa C-末端结构域的优化蛋白制备物，该蛋白是与N端His _（6）标签。所有重组VacA构建体均以不溶性包裹体形式在大肠杆菌中过表达，当磷酸盐缓冲液（pH 7.4）补充5-6 M尿素时，它们是可溶的。固定后-Ni ²⁺在5-M尿素变性条件下进行亲和纯化，始终获得55 / 59-kDa结构域的同质产物（纯度> 95％），而两个成熟VacA变体和33-kDa截短产物的纯度仅为〜80％，因此需要通过尺寸排阻色谱法进一步纯化。连续的重新折叠后通过通过优化的分步透析，已证实所有重新折叠的VacA蛋白都具有针对培养的人胃上皮细胞的细胞毒和空泡活性，尽管观察到的VacA-m2活性低于m1型变体。本文所述的优化方案可有效地大量生产高纯度重组VacA蛋白（每升培养物约30-40 mg），这将为进一步研究不同VacA变体的序列结构和功能关系铺平道路。