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Selective Isolation of Myosin Subfragment-1 with a DNA-Polyoxovanadate Bioconjugate
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2017-12-07 00:00:00 , DOI: 10.1021/acs.bioconjchem.7b00597
Qing Chen 1 , Xue Hu 1 , Dan-Dan Zhang 1 , Xu-Wei Chen 1 , Jian-Hua Wang 1
Affiliation  

The bioconjugation of a polyoxometalate (POMs), i.e., dodecavanadate (V12O32), to DNA strands produces a functional labeled DNA primer, V12O32-DNA. The grafting of DNA primer onto streptavidin-coated magnetic nanoparticles (SVM) produces a novel composite, V12O32[email protected] The high binding-affinity of V12O32 with the ATP binding site in myosin subfragment-1 (S1) facilitates favorable adsorption of myosin, with an efficiency of 99.4% when processing 0.1 mL myosin solution (100 μg mL–1) using 0.1 mg composite. Myosin adsorption fits the Langmuir model, corresponding to a theoretical adsorption capacity of 613.5 mg g–1. The retained myosin is readily recovered by 1% SDS (m/m), giving rise to a recovery of 58.7%. No conformational change is observed for myosin after eliminating SDS by ultrafiltration. For practical use, high-purity myosin S1 is obtained by separation of myosin from the rough protein extract from porcine left ventricle, followed by digestion with α-chymotryptic and further isolation of S1 subfragment. The purified myosin S1 is identified with matrix-assisted laser desorption/ionization time-of-flight/mass spectrometry, giving rise to a sequence coverage of 38%.

中文翻译:

DNA-聚氧钒酸盐生物共轭物对肌球蛋白亚片段1的选择性分离

多金属氧酸盐(POM),即十二烷酸盐(V 12 O 32)与DNA链的生物缀合产生功能性标记的DNA引物V 12 O 32 -DNA 。将DNA引物嫁接到抗生蛋白链菌素包被的磁性纳米颗粒(SVM)上产生了一种新型复合物V 12 O 32 [电子邮件保护] V 12 O 32与肌球蛋白亚片段1(S1)中的ATP结合位点具有高结合亲和力促进了肌球蛋白的良好吸附,在处理0.1 mL肌球蛋白溶液(100μgmL –1)时效率为99.4%)使用0.1毫克复合材料。肌球蛋白的吸附符合Langmuir模型,对应的理论吸附容量为613.5 mg g –1。残留的肌球蛋白很容易通过1%SDS(m / m)回收,回收率为58.7%。通过超滤消除SDS后,没有观察到肌球蛋白的构象变化。对于实际应用,高纯度的肌球蛋白S1是通过从猪左心室的粗蛋白提取物中分离肌球蛋白,然后用α-胰凝乳蛋白酶消化并进一步分离S1亚片段而获得的。通过基质辅助激光解吸/电离飞行时间/质谱法鉴定纯化的肌球蛋白S1,序列覆盖率达到38%。
更新日期:2017-12-07
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