Expression and Activation of Horseradish Peroxidase&-Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes
Expression and Activation of Horseradish Peroxidase&-Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes
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辣根过氧化物酶的表达和激活
DOI：
10.1002/biot.201700624
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发表时间：
2018
期刊：
影响因子：
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通讯作者：
N. Kamiya
中科院分区：
文献类型：
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作者：
Patma;K. Minamihata;T. Tatsuke;J. M. Lee;T. Kusakabe;N. Kamiya
重组蛋白生产可以通过对目标蛋白进行遗传修饰来产生具有所需功能的人造蛋白。辣根过氧化物酶(HRP)作为一种报告酶在生物技术中得到了广泛的应用，然而，HRP的重组生产并不是很成功，这阻碍了HRP的基因修饰。包含抗体结合蛋白和HRP的融合蛋白将是基于HRP的高质量诊断系统的理想生物探针。设计了一种HRP-蛋白A/G融合蛋白(HRP-PAg)，并首次对其在家蚕(Bombyx Mori)中的生产进行了评价。HRP-PAg以可溶性载脂蛋白的形式表达，与氯化高铁血红素孵育后可被成功激活。将活化的辣根过氧化物酶-PAg直接用于酶联免疫吸附试验，在4 °C下保持其活性20天以上，并通过微生物转谷氨酰胺酶反应将其生物素修饰。生物素化的HRP-PAg与链霉亲和素偶联形成HRP-PAg多聚体，多聚体HRP-PAg在ELISA体系中产生比单体HRP-PAg更高的信号。重组HRP在家蚕中的成功生产将有助于创造新的基于HRP的生物结合物，并通过应用酶后翻译后修饰来进一步实现HRP的功能化。
Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio‐probe for high‐quality HRP‐based diagnostic systems. A HRP‐protein A/G fusion protein (HRP‐pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP‐pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP‐pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP‐pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP‐pAG is conjugated with streptavidin to form a HRP‐pAG multimer and the multimeric HRP‐pAG produced higher signals in the ELISA system than monomeric HRP‐pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP‐based bioconjugates as well as further functionalization of HRP by applying enzymatic post‐translational modifications.