Expression and simple, one-step purification of fragile histidine triad (Fhit) tumor suppressor mutant forms in Escherichia coli and their interaction with protoporphyrin IX
Expression and simple, one-step purification of fragile histidine triad (Fhit) tumor suppressor mutant forms in Escherichia coli and their interaction with protoporphyrin IX
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DOI：
10.1007/s10529-007-9322-9
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发表时间：
2007-02
影响因子：
2.7
通讯作者：
J. Zawacka-Pankau;A. Podhajska
中科院分区：
文献类型：
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作者：
J. Zawacka-Pankau;A. Podhajska
人类脆性组氨酸三联体（FHIT）基因的产物是一种肿瘤抑制蛋白，其细胞背景仍很大程度上未知。我们之前已经证明它与原卟啉 IX（一种光敏剂）结合，从而改变其体外酶活性。 Fhit，三磷酸二腺苷 (Ap3A) 水解酶，具有组氨酸三联体 His-φ-His-φ-His-φφ 的活性位点。所谓的组氨酸 Fhit 突变体（His94Asn、His96Asn 和 His98Asn）在体外表现出高度降低的活性，然而，其抗肿瘤功能尚未得到充分描述。在这项工作中，我们将组氨酸突变体的 cDNA 克隆到 pPROEX-1 载体中，从而产生 His6 融合蛋白。突变蛋白：Fhit-H94N、Fhit-H96N 和 Fhit-H98N 在大肠杆菌BL21(DE3) 中表达，并通过改进的一步式 Ni-次氮基三乙酸酯树脂亲和层析纯化（高达 95%）。最终产量为 1 g 细菌（湿重）2 mg 均质蛋白质。通过先前描述的测定法评估纯化蛋白质的活性。相同的纯化程序产生了 0.8 mg/ml 的高活性野生型 Fhit 蛋白（Ap3A 的 Km 值为 5.7 μM）。重要的是，纯化的 Fhit 突变体在体外也与光敏剂原卟啉 IX 相互作用。
The product of humanfragile histidine triad(FHIT) gene is a tumor suppressor protein of still largely unknown cellular background. We have shown previously that it binds protoporphyrin IX (a photosensitizer) which alters its enzymatic activity in vitro. Fhit, diadenosine triphosphate (Ap3A) hydrolase, possesses the active site with histidine triad His-φ-His-φ-His-φφ. So-called histidine Fhit mutants (His94Asn, His96Asn and His98Asn) exhibit highly reduced activity in vitro, however, their antitumor function has not been fully described yet. In this work we have cloned the cDNAs of histidine mutants into pPROEX-1 vector allowing the production of His6-fusion proteins. The mutated proteins: Fhit-H94N, Fhit-H96N and Fhit-H98N, were expressed inEscherichia coliBL21(DE3) and purified (up to 95%) by an improved, one-step affinity chromatography on Ni-nitrilotriacetate resin. The final yield was 2 mg homogenous proteins from 1 g bacteria (wet wt). The activity of purified proteins was assessed by previously described assay. The same purification procedure yielded 0.8 mg/ml and highly active wild-type Fhit protein (Kmvalue for Ap3A of 5.7 μM). Importantly, purified mutant forms of Fhit also interact with a photosensitizer, protoporphyrin IX in vitro.