Protocols and Applications Guide (US Letter Size) - Promega

||||| 5Protein Expression
PN060 Applications of the TNT® T7 Quick
Coupled Transcription/Translation System
(www.promega.com
/pnotes/60/6079_07/promega.html)
PN066 Application of the TNT® T7 Quick System
to Selection and Evolution of Antibody
Combining Sites
(www.promega.com
/pnotes/66/7014_14/7014_14.html)
PN067 In vitro Expression Cloning Using the
TNT® Coupled Reticulocyte Lysate System
(www.promega.com
/pnotes/67/7201_02/7201_02.html)
PN070 Applications of Promega's In Vitro
Expression Systems
(www.promega.com
/pnotes/70/7618_02/7618_02.html)
PN081 Express More Functional Protein: TNT®
Quick Coupled Transcription/Translation
Systems
(www.promega.com
/pnotes/81/9939_16/9939_16.html)
PN088 Technically Speaking: TNT® Rabbit
Reticulocyte Lysate Systems–Easy Protein
Expression
(www.promega.com
/pnotes/88/12162_24/12162_24.html)
PN093 TNT® SP6 High-Yield Protein Expression
System: More Protein from a Coupled
Transcription/Translation System
(www.promega.com
/pnotes/93/14023_15/14023_15.html)
PN100 Cell-Free Protein Expression with the
TNT® T7 Insect Cell Extract Protein
Expression System
(www.promega.com
/pnotes/100/16620_11/16620_11.html)
Citations
Nagase , T. et al. (2008) Exploration of human ORFeome:
High-throughput preparation of ORF clones and efficient
characterization of their protein products. DNA Research
15, 137–149..
The authors created HaloTag® fusion proteins and
examined expression of these proteins in vitro and in COS7
and HEK293 cells. They also performed comparisons
between the Flexi® System and Gateway® cloning system,
specifically examining the effects of flanking sequences on
protein expression in in vitro translation systems including
the SP6 High-Yield Protein Expression System. They
confirmed that the cellular localization of the HaloTag®
fusion proteins was consistent with results obtained using
GFP-fusions.
PubMed Number: 18316326
Protocols & Applications Guide
www.promega.com
rev. 6/09
Citations
Muik, M. et al. (2008) Dynamic coupling of the putative
coiled-coil domain of ORAI1 with STIM1 mediates ORAI1
channel activation. J. Biol. Chem. 283, 8014–8022. .
The authors performed protein pull-down assays to
characterize the interaction of ORAI1 and STIM1, two
protein components of the calcium-release calcium current.
His6-STIM1 C terminus and ORAI1 were synthesized using
the TNT® Coupled Reticulocyte Lysate System in the
presence of 35S, and His6-STIM1 C terminus was
immobilized using MagZ Binding Particles. An aliquot
of the TNT® reaction expressing ORAI1 was added to the
particles, and proteins were washed, eluted using increasing
concentrations of imidazole (10–40mM) and analyzed by
SDS-PAGE. In a second set of pull-down assays,
His6-STIM1 C terminus was used to pull down ORA1 Nand
C-terminal fragments expressed as GST fusion proteins.
The His6-STIM1 C terminus protein was purified from
transiently transfected HEK293 cells using the MagneHis
Protein Purification System.
PubMed Number: 18187424
IV. Rabbit Reticulocyte Lysate Translation Systems
A. Standard Rabbit Reticulocyte Lysate Translation
The Rabbit Reticulocyte Lysate Translation System plays
an important role in characterization of mRNA translation
products, investigation of transcriptional and translational
control, and co-translational processing of secreted proteins
by the addition of microsomal membranes to the translation
reaction. Rabbit Reticulocyte Lysate is prepared from New
Zealand white rabbits injected with phenylhydrazine using
a standard protocol to increase reticulocyte production
(Pelham and Jackson, 1976). The reticulocytes are harvested,
and any contaminating cells that could otherwise alter the
translational properties of the final extract are removed.
After lysis of the reticulocytes, the extract is treated with
micrococcal nuclease to digest endogenous mRNA and
thus reduce background translation to a minimum. The
lysate contains the cellular components necessary for
protein synthesis: tRNA, ribosomes, amino acids, and
initiation, elongation and termination factors. Reticulocyte
Lysate is further optimized for mRNA translation by adding
several supplements as described in Section I.A.
Rabbit reticulocyte lysate has been reported to contain a
variety of post-translational processing activities, including
acetylation, isoprenylation, proteolysis and some
phosphorylation activity (Glass and Pollard, 1990).
Processing events such as signal peptide cleavage and core
glycosylation can be examined by adding canine
microsomal membranes to a translation reaction (Andrews,
1987; Walter and Blobel, 1983; Thompson and Beckler, 1992)
B. Flexi® Rabbit Reticulocyte System—In Vitro Translation
The Flexi® Rabbit Reticulocyte Lysate System allows greater
flexibility of reaction conditions than the standard Rabbit
Reticulocyte Lysate System, Nuclease Treated. Different
PROTOCOLS & APPLICATIONS GUIDE 5-8