Cloning, expression, purification and biochemical characterization of recombinant metallothionein from the white shrimp Litopenaeus vannamei
| Autor: | Monserrath Felix-Portillo, Rosa O. Méndez-Estrada, Miguel Ángel Martínez-Téllez, Gloria Yepiz-Plascencia, Jorge Duarte-Gutiérrez, Lilia Leyva-Carrillo |
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| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
Recombinant Fusion Proteins Genetic Vectors Chitin 01 natural sciences Chromatography Affinity 03 medical and health sciences chemistry.chemical_compound Affinity chromatography Penaeidae Chitin binding 010608 biotechnology Escherichia coli Metallothionein Animals Cloning Molecular 030304 developmental biology chemistry.chemical_classification 0303 health sciences Reactive oxygen species Fusion protein Shrimp Biochemistry chemistry Trolox Intein Copper Biotechnology Cadmium |
| Zdroj: | Protein expression and purification. 166 |
| ISSN: | 1096-0279 |
| Popis: | Metallothioneins (MTs) are cysteine rich proteins with antioxidant capacity that participate in the homeostasis and detoxification of metals and other cellular processes, and help to counteract the oxidative stress produced by Reactive Oxygen Species (ROS). The production of ROS increases during several stress conditions, including metal intoxication and hypoxia (oxygen deficiency). During hypoxia the expression of the MT gene is induced in the shrimp Litopenaeus vannamei; however, the MT protein coded by this gene has not been purified nor characterized. In this work, the coding sequence of L. vannamei MT was cloned and overexpressed in Escherichia coli as a fusion protein, containing an intein and a chitin binding domain (CBD). The MT was purified by chitin affinity chromatography and its antioxidant capacity and ability to bind cadmium (Cd) and copper (Cu) were evaluated. This MT has an antioxidant capacity of 27.23 μM equivalent to Trolox in a 100 μg/mL solution. Addition of CdCl2 to the culture media augments 273-fold the Cd content, while addition of CuCl2 increases Cu content 569-fold in the purified MT. Thus, the shrimp MT gene codes for a functional protein that has antioxidant capacity and binds Cu and Cd. |
| Databáze: | OpenAIRE |
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