Cloning, expression, purification, and characterization of biologically active recombinant hemagglutinin-33, type A botulinum neurotoxin associated protein
Cloning, expression, purification, and characterization of biologically active recombinant hemagglutinin-33, type A botulinum neurotoxin associated protein
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DOI：
10.1007/s10930-006-9041-4
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发表时间：
2007-01-01
期刊：
影响因子：
3
通讯作者：
Singh, Bal Ram
中科院分区：
文献类型：
--
作者：
Zhou, Yu;Paturi, Sowmya;Singh, Bal Ram
A型肉毒杆菌神经毒素是人类已知的毒性最大的物质，是由A型肉毒杆菌与一组神经毒素相关蛋白（nap）通过聚类基因的多顺反性表达产生的复合物。血凝素-33 （Hn-33）是nap的一种33 kDa的亚组分，它对蛋白酶消化具有抗性，这一特征可能与保护肉毒杆菌神经毒素免受蛋白质水解有关。为了充分了解Hn-33的功能，在不考虑生物安全风险的情况下，需要大量的Hn-33来培养肉毒杆菌。肉毒杆菌产生的高A + T含量基因克隆困难。本研究首次利用Gateway技术克隆了在大肠杆菌中表达的功能性Hn-33。重组Hn-33的产量约为每升大肠杆菌培养12毫克。从圆二色光谱可以看出，重组Hn-33在水溶液中折叠良好，抗温度变性，尽管在分子表面存在裂解位点，但完全抵抗胰蛋白酶蛋白水解，并保持与天然Hn-33血凝反应相当的生物活性。
Botulinum neurotoxin type A, the most toxic substance known to mankind, is produced by Clostridium botulinum type A as a complex with a group of neurotoxin-associated proteins (NAPs) through polycistronic expression of a clustered group of genes. Hemagglutinin-33 (Hn-33) is a 33 kDa subcomponent of NAPs, which is resistant to protease digestion, a feature likely to be involved in the protection of the botulinum neurotoxin from proteolysis. In order to fully understand the function of Hn-33, large amounts of Hn-33 will be needed without dealing with biosafety risks to grow large cultures of C. botulinum. There are difficulties to clone the genes with the high A + T contents produced by C. botulinum. We report here for the first time using the Gateway technology to clone functional Hn-33 that has been expressed in E. coli. The yield of the recombinant Hn-33 was about 12 mg per liter of E. coli culture. The recombinant Hn-33 folds well in aqueous solution as shown with circular dichroism spectra, resists temperature-denaturation, is totally resistant to trypsin proteolysis despite the presence of cleavage sites on the molecular surface, and maintains its biological activities comparable to the native Hn-33 hemagglutination.