Rapid production of pure recombinant actin isoforms in Pichia pastoris
Actins are major eukaryotic cytoskeletal proteins, and they are involved in many important cell functions, including cell division, cell polarity, wound healing and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively for biochemical studies of t...
| Autores: | , , , , , , , , , , , , , , , |
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| Tipo de recurso: | artículo |
| Fecha de publicación: | 2018 |
| País: | España |
| Institución: | Universidad de La Laguna (ULL) |
| Repositorio: | RIULL. Repositorio Institucional de la Universidad de La Laguna |
| OAI Identifier: | oai:riull.ull.es:915/38849 |
| Acceso en línea: | http://riull.ull.es/xmlui/handle/915/38849 |
| Access Level: | acceso abierto |
| Palabra clave: | Actin Actin purification Biochemistry Cytoskeleton |
| Sumario: | Actins are major eukaryotic cytoskeletal proteins, and they are involved in many important cell functions, including cell division, cell polarity, wound healing and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively for biochemical studies of the non-muscle actin cytoskeleton. Here, we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris. Actin is expressed as a fusion with the actin-binding protein thymosin β4 and purified by means of an affinity tag introduced in the fusion. Following cleavage of thymosin β4andtheaffinity tag, highly purified functional full-length actin is liberated. We purify actins from Saccharomyces cerevisiae and Schizosaccharomyces pombe, and the β- and γ-isoforms of human actin. We also report a modification of the method that facilitates expression and purification of arginylated actin, a form of actin thought to regulate dendritic actin networks in mammalian cells. The methods we describe can be performed in all laboratories equipped for molecular biology, and should greatly facilitate biochemical and cell biological studies of the actin cytoskeleton. |
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