Protocols and Applications Guide (US Letter Size) - Promega

||||| 5Protein Expression
I. Introduction
Cell-free systems for in vitro gene expression and protein
synthesis have been described for many different
prokaryotic (Zubay, 1973) and eukaryotic (Pelham and
Jackson, 1976; Anderson et al. 1983) systems. Eukaryotic
cell-free systems, such as rabbit reticulocyte lysate and
wheat germ extract, are prepared from crude extract
containing all the components required for translation of
in vitro-transcribed RNA templates. Eukaryotic cell-free
systems use isolated RNA synthesized in vivo or in vitro
as a template for the translation reaction (e.g., Rabbit
Reticulocyte Lysate Systems [Cat.# L4960, L4540] or Wheat
Germ Extract Systems [Cat.# L4380]). Coupled eukaryotic
cell-free systems combine a prokaryotic phage RNA
polymerase with eukaryotic extracts and utilize an
exogenous DNA or PCR-generated templates with a phage
promoter for in vitro protein synthesis (Figure 5.1) ( TNT®
Coupled Reticulocyte Lysate [Cat.# L4600, L4610, L4950,
L5010, L5020], TNT® Quick Coupled
Transcription/Translation Systems [Cat.# L1170, L2080],
TNT® T7 Quick for PCR DNA [Cat.# L5540] and TNT®
Wheat Germ Extract Systems [Cat.# L4120, L4130, L4140,
L5030, L5040]).
Proteins translated using the TNT® Coupled Systems can
be used in many types of functional studies. TNT® Coupled
Transcription/Translation reactions have traditionally been
used to confirm open reading frames, study protein
mutations and make proteins in vitro for protein:DNA
binding studies, protein activity assays, or protein:protein
interaction studies. Recently, proteins expressed using the
TNT® Coupled Systems have also been used in assays to
confirm yeast two-hybrid interactions, perform in vitro
expression cloning (IVEC) and make protein substrates for
enzyme activity or protein modification assays. For a listing
of recent citations using the TNT® Coupled Systems in
various applications, please visit:
www.promega.com/citations/ (www.promega.com
/citations)
Transcription and translation are typically coupled in
prokaryotic systems; that is, they contain an endogenous
or phage RNA polymerase, which transcribes mRNA from
an exogenous DNA template. This RNA is then used as a
template for translation. The DNA template may be either
a gene cloned into a plasmid vector (cDNA) or a PCR
generated template. A ribosome binding site (RBS) is
required for templates translated in prokaryotic systems.
During transcription, the 5´-end of the mRNA becomes
available for ribosome binding and translation initiation,
allowing transcription and translation to occur
simultaneously. Prokaryotic systems are available that use
DNA templates containing either prokaryotic promoters
(such as lac or tac; E. coli S30 Extract System for Circular
and Linear DNA [Cat.# L1020 and L1030] or a phage RNA
polymerase promoter; E. coli T7 S30 Extract System for
Circular DNA [Cat.# L1130]).
Protocols & Applications Guide
www.promega.com
rev. 6/09
Most in vitro systems produce picomole (or nanogram)
amounts of proteins per 50µl reaction. This yield is usually
sufficient for most types of radioactive, fluorescent and
antibody analyses, such as polyacrylamide gel separation,
Western blotting, immunoprecipitation and/or, depending
on the protein of interest, enzymatic or biological activity
assays. For radioactive detection, a radioactive amino acid
is added to the translation reaction and, after incorporation,
the gene product is identified by autoradiography following
SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
Alternatively, non-radioactive labeling methods such as
fluorescent, chemiluminescent or colorimetric detection
may be used (i.e., Transcend and FluoroTect Systems;
Sections VIII and IX, respectively). If antibodies to the
protein are available, then techniques such as
immunoblotting or immunoprecipitation can be used. The
functional activity of in vitro-translated products can often
be detected directly in the reaction mixture. If protein
purification is necessary, fusion of the protein to a
purification “tag” allows the protein to be isolated from
the in vitro translation reaction and subsequently studied.
Since protein synthesis reactions can be driven by RNA
templates (translation; Section I.A) or DNA templates
(coupled transcription/translation; Section I.B), the type of
template is generally the first consideration when choosing
an appropriate system. Promega translation and coupled
transcription/translation systems are summarized in Tables
5.1 and 5.2, respectively. All systems provide reliable,
convenient and efficient methods to initiate translation and
produce full-size protein products. For assistance in
choosing a cell-free protein expression system, the Cell-Free
Protein Expression Product Selector (www.promega.com
/selectors/tnt/) is available.
Cell-free protein synthesis systems have become standard
tools for the in vitro expression of proteins from cloned
genes. Applications for in vitro expression systems include
analysis of protein:protein (/multimedia/tnt01.htm
(www.promega.com/multimedia/tnt01.htm)) and
protein:nucleic acid interactions (/multimedia/tnt02.htm
(www.promega.com/multimedia/tnt02.htm)). In addition,
these systems can be used for mutational analysis, epitope
mapping, in vitro evolutionary studies, protein truncation
test (PTT) (Powell et al. 1993; Roest et al. 1993), clone
verification, functional analysis, mutagenesis and domain
mapping, ribosome display (Mattheakis et al. 1994; Hanes
and Pluckthun, 1997) and in vitro expression cloning (IVEC)
(Lustig et al. 1997; King et al. 1997), molecular diagnostics
and high-throughput screening (Novac et al. 2004). In vitro
expression systems also offer significant time savings over
in vivo systems. The primary advantage of in vitro
translation over in vivo protein expression is that in vitro
systems allow the use of a defined template to direct protein
synthesis. In vitro systems also have the ability to express
toxic, proteolytically sensitive, or unstable gene products,
and allow the specific labeling of gene products so that
individual proteins can be monitored in complex reaction
mixtures.
PROTOCOLS & APPLICATIONS GUIDE 5-1