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2014 ; 108
(ä): 16.31.1-16.31.22
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Protein synthesis using a reconstituted cell-free system
#MMPMID25271715
Tuckey C
; Asahara H
; Zhou Y
; Chong S
Curr Protoc Mol Biol
2014[Oct]; 108
(ä): 16.31.1-16.31.22
PMID25271715
show ga
Most cell-free protein-synthesis systems are based on cell extracts, which often
contain undesirable activities. Reconstituted systems, by contrast, are composed
of a defined number of purified and recombinant components with minimal nuclease
and protease activities. This unit describes the use of a particular commercial
reconstituted system, PURExpress. This system allows in vitro synthesis of
proteins from mRNA and circular and linear DNA templates, as well as
co-translational labeling of proteins. Unique to this system, all recombinant
protein components of the system are His-tagged, allowing purification of the
synthesized untagged protein by removing the rest of the system's components.
Newly synthesized proteins can often be visible on an SDS-PAGE gel and directly
assayed for their functions without labeling and purification. Certain components
of the system, such as ribosomes or release factors, can be omitted for specific
applications. Such "delta" versions of the system are well suited for studies of
bacterial translation, assays of ribosome function, incorporation of unnatural
amino acids, and ribosome display of protein libraries.
free
free
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