Obtaining Soluble Folded Proteins from Inclusion Bodies Using Sarkosyl, Triton X-100, and CHAPS: Application to LB and M9 Minimal Media.
Curr Protoc Protein Sci
; 84: 6.13.1-6.13.24, 2016 Apr 01.
Article
em En
| MEDLINE
| ID: mdl-27038270
ABSTRACT
This unit describes a straightforward and efficient method of using sarkosyl to solubilize and recover difficult recombinant proteins, such as GST- and His6 -tagged fusion proteins, that are overexpressed in E. coli. This protocol is especially useful for rescuing recombinant proteins overexpressed in M9 minimal medium. Sarkosyl added to lysis buffers helps with both protein solubility and cell lysis. Higher percentage sarkosyl (up to 10%) can extract >95% of soluble protein from inclusion bodies. In the case of sarkosyl-solubilized GST-fusion proteins, batch-mode affinity purification requires addition of a specific ratio of Triton X-100 and CHAPS, while sarkosyl-solubilized His6 -tagged fusion proteins can be directly purified on Ni(2+) resin columns. Proteins purified by this method could be widely used in biological assays, structure analysis and mass spectrum assay.
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Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Sarcosina
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Proteínas Recombinantes de Fusão
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Corpos de Inclusão
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Ácidos Cólicos
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Octoxinol
Idioma:
En
Ano de publicação:
2016
Tipo de documento:
Article