Cyclophilins are highly conserved proteins associated with peptidyl-prolyl cis-trans isomerase activity (PPIase). The present study was designed to analyze the biological activity of recombinant cyclophilin from the marine red algae Pyropia yezoensis (PyCyp). The cyclophilin gene from P. yezoensis was cloned into the pPROEX-HTA expression vector. The plasmid was transformed into BL21 Escherichia coli by high efficiency transformation. Recombinant protein was expressed using 0.1 mM IPTG and the fusion protein was purified by affinity column chromatography. The His-tag was removed by TEV protease. The recombinant protein was further purified on a HiPrep Sephacryl S-200 HR column and by reversed-phase high performance liquid chromatography with a Sep-pak plus C18 column. Purified cyclophilin was characterized by a variety of analytical methods and analyzed for its peptidyl-prolyl isomerase activity. Our recombinant PyCyp was shown to catalyze cis-trans isomerization. PyCyp was also evaluated for antimicrobial activity against both Gram-positive and Gram-negative bacteria cultures and showed significant antibacterial activity against tested pathogens. PyCyp was shown to permeabilize bacterial membranes as evidenced by increased fluorescence intensity in SYTOX Green uptake assays with Staphylococcus aureus. The radical scavenging activity of PyCyp increased in a dose-dependent manner, indicating significant antioxidant activity. This study provides information for the development of therapeutic proteins from marine algae.

中文翻译：

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肽基脯氨酰异构酶和紫斑拟南芥（PyCyp）重组蛋白亲环蛋白的生物学活性。

亲环蛋白是与肽基-脯氨酰顺反异构酶活性（PPIase）相关的高度保守的蛋白。本研究旨在分析来自海洋红藻Pyopia yezoensis（PyCyp）的重组亲环蛋白的生物学活性。将来自耶氏疟原虫的亲环蛋白基因克隆到pPROEX-HTA表达载体中。通过高效转化将质粒转化到BL21大肠杆菌中。使用0.1 mM IPTG表达重组蛋白，并通过亲和柱色谱法纯化融合蛋白。His标签被TEV蛋白酶去除。重组蛋白进一步在HiPrep Sephacryl S-200 HR色谱柱上纯化，并通过高效液相色谱（Sep-pak plus C18色谱柱）进行反相纯化。通过多种分析方法对纯化的亲环蛋白进行表征，并分析其肽基-脯氨酰异构酶活性。我们的重组PyCyp被证明可以催化顺反异构化。还评估了PyCyp对革兰氏阳性和革兰氏阴性细菌培养物的抗菌活性，并显示出对测试病原体的显着抗菌活性。PyCyp被证明可以渗透细菌膜，这在金黄色葡萄球菌的SYTOX Green吸收试验中荧光强度的增加得到了证明。PyCyp的自由基清除活性以剂量依赖性方式增加，表明具有显着的抗氧化剂活性。这项研究为开发来自海藻的治疗性蛋白质提供了信息。我们的重组PyCyp被证明可以催化顺反异构化。还评估了PyCyp对革兰氏阳性和革兰氏阴性细菌培养物的抗菌活性，并显示出对测试病原体的显着抗菌活性。PyCyp被证明可以渗透细菌膜，这在金黄色葡萄球菌的SYTOX Green吸收试验中荧光强度的增加得到了证明。PyCyp的自由基清除活性以剂量依赖性方式增加，表明具有显着的抗氧化剂活性。这项研究为开发来自海藻的治疗性蛋白质提供了信息。我们的重组PyCyp被证明可以催化顺反异构化。还评估了PyCyp对革兰氏阳性和革兰氏阴性细菌培养物的抗菌活性，并显示出对测试病原体的显着抗菌活性。PyCyp被证明可以渗透细菌膜，这在金黄色葡萄球菌的SYTOX Green吸收试验中荧光强度的增加得到了证明。PyCyp的自由基清除活性以剂量依赖性方式增加，表明具有显着的抗氧化剂活性。这项研究为开发来自海藻的治疗性蛋白质提供了信息。PyCyp被证明可以渗透细菌膜，这在金黄色葡萄球菌的SYTOX Green吸收试验中荧光强度的增加得到了证明。PyCyp的自由基清除活性以剂量依赖性方式增加，表明具有显着的抗氧化剂活性。这项研究为开发来自海藻的治疗性蛋白质提供了信息。PyCyp被证明可以渗透细菌膜，这在金黄色葡萄球菌的SYTOX Green吸收试验中荧光强度的增加得到了证明。PyCyp的自由基清除活性以剂量依赖性方式增加，表明具有显着的抗氧化剂活性。这项研究为开发来自海藻的治疗性蛋白质提供了信息。