Expression in Escherichia coli and simple purification of human Fhit protein.
Expression in Escherichia coli and simple purification of human Fhit protein.
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DOI：
10.1006/prep.1999.1199
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发表时间：
2000-04
影响因子：
1.6
通讯作者：
T. Pawełczyk;R. Kowara;F. Golebiowski;A. Matecki
中科院分区：
文献类型：
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作者：
T. Pawełczyk;R. Kowara;F. Golebiowski;A. Matecki
脆性组氨酸三联体(FHIT)蛋白是一种具有二腺苷5‘，5“’-P(1)，P(3)-三磷酸(AP(3)A)不对称水解酶活性的同源二聚体蛋白。我们已将人FHIT基因克隆到pPROEX-1载体中，并在大肠杆菌中高效表达，序列为Met-Gly-Hly(6)-Asp-Tyr-Asp-Ile-Pro-Thr-Thr，其N端融合了rTEV蛋白酶切割位点H6TV。H6TV-FHIT在BL21(DE3)细胞中37℃表达3h，从1个L细胞培养物(约4g细胞)中产生30 mg H6TV-FHIT。以硝酸镍树脂和咪唑缓冲液为洗脱剂，一步纯化H6TV-FHIT蛋白，得率为80%。H6TV-FHIT与rTEV蛋白酶在4℃下孵育24小时，H6TV多肽被完全切割。没有非特异性的切割产物。纯化的FHIT蛋白可以在4℃下保存3周而不失去活性。当在含有25%甘油的缓冲液中储存时，纯蛋白质在-20℃下至少稳定18个月。纯化的FHIT具有很高的活性，AP(3)A的K(M)值为0.9微米，单体的k(CAT)值为7.2+/-1.6 S(-1)(n=5)。未结合的FHIT蛋白与H6TV-FHIT融合蛋白的催化性质基本相同。这表明含有6个组氨酸的24位氨基酸与FHIT的N端融合，不干扰活性同源二聚体的形成或AP(3)A的结合。
The fragile histidine triad (Fhit) protein is a homodimeric protein with diadenosine 5',5"'-P(1),P(3)-triphosphate (Ap(3)A) asymmetrical hydrolase activity. We have cloned the human cDNA Fhit in the pPROEX-1 vector and expressed with high yield in Escherichia coli with the sequence Met-Gly-His(6)-Asp-Tyr-Asp-Ile-Pro-Thr-Thr followed by a rTEV protease cleavage site, denoted as "H6TV," fused to the N-terminus of Fhit. Expression of H6TV-Fhit in BL21(DE3) cells for 3 h at 37 degrees C produced 30 mg of H6TV-Fhit from 1 L of cell culture ( approximately 4 g of cells). The H6TV-Fhit protein was purified to homogeneity in a single step, with a yield of 80%, using nickel-nitrilotriacetate resin and imidazole buffer as eluting agent. Incubation of H6TV-Fhit with rTEV protease at 4 degrees C for 24 h resulted in complete cleavage of the H6TV peptide. There were no unspecific cleavage products. The purified Fhit protein could be stored for 3 weeks at 4 degrees C without loss of activity. The pure protein was stable at -20 degrees C for at least 18 months when stored in buffer containing 25% glycerol. Purified Fhit was highly active, with a K(m) value for Ap(3)A of 0.9 microM and a k(cat)(monomer) value of 7.2 +/- 1.6 s(-1) (n = 5). The catalytic properties of unconjugated Fhit protein and the H6TV-Fhit fusion protein were essentially identical. This indicates that the 24-amino-acid peptide containing the six histidines fused to the N-terminus of Fhit does not interfere in forming the active homodimers or in the binding of Ap(3)A.