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A self-catalyzing strategy for co-immobilization of two distinct proteins at equimolar ratio: A case study of 3A and 2C to develop a chromatographic method for finding prospective dual-target compoundsfrom complex matrices.
Quan, Jia; Ou, Yuanyuan; Long, Kaihua; Li, Yu; Kang, Jing; Wang, Yaqi; Zhao, Xue; Zhao, Xinfeng.
Afiliación
  • Quan J; Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an, 710069, China.
  • Ou Y; Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an, 710069, China.
  • Long K; Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an, 710069, China.
  • Li Y; Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an, 710069, China.
  • Kang J; Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an, 710069, China.
  • Wang Y; Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an, 710069, China.
  • Zhao X; Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an, 710069, China. Electronic address: xxuezhao@nwu.edu.cn.
  • Zhao X; Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an, 710069, China. Electronic address: zhaoxf@nwu.edu.cn.
Anal Chim Acta ; 1318: 342950, 2024 Aug 22.
Article en En | MEDLINE | ID: mdl-39067927
ABSTRACT

BACKGROUND:

Immobilized proteins hold promise as the basic units that have enabled a broad range of analytical applications within chemical measurement science. As yet, the co-immobilization of diverse proteins at precise ratio and whether they give rise to improved analytical performance remain challengeable. Herein, we utilized a circularly permuted HaloTag (cpHaloTag) to achieve the co-immobilization of two proteins at precise ratio, which was applied in developing a chromatographic method with improved specificity for pursuing dual-target compounds.

RESULTS:

The methodology involved the fusion 3A and 2C at N- and C-terminuses of cpHaloTag, the immobilization of the fusion protein onto silica gel through bioorthogonal reaction, the morphological and functional characterization, the application in finding dual-target compounds. Expression of the fusion protein in E. coli system showed a yield of milligram level with the presence of 3A and 2C domains. Immobilization of the protein was achieved in 10 min with a reaction efficiency more than 88.5 %. Immobilized 3A-cpHalo-2C exhibited higher specificity and better retentions of canonical compounds of the two enzymes in comparison with the column containing immobilized 3A or 2C alone. In real sample application, screening analysis found that hyperoside, cymaroside, and baicalin were dual-target compounds in concert with 3A and 2C in Shuanghuanglian extract.

SIGNIFICANCE:

Taking 3A and 2C as probe, we proposed a simple method for direct co-immobilization of diverse proteins from cell lysates and demonstrated an affinity chromatographic-based dual-target compound screening platform. The implications of these methodology are possible to insight the de novo design of multi-target surface for fabricating new bioanalytical methods with improved performance.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Enzimas Inmovilizadas Idioma: En Revista: Anal Chim Acta Año: 2024 Tipo del documento: Article País de afiliación: China Pais de publicación: Países Bajos
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Enzimas Inmovilizadas Idioma: En Revista: Anal Chim Acta Año: 2024 Tipo del documento: Article País de afiliación: China Pais de publicación: Países Bajos