The Role of Cell-Free Rabbit Reticulocyte Expression ... - Promega

The Role of Cell-Free Rabbit Reticulocyte Expression Systems in Functional Proteomics
Figure 3. �rotein:�NA fusion. Co���ent �NA:protein �o�p�exes ��n �e gener�ted �y �ig�tion
of � �NA:puro�y�in �inker to the in �itro tr�ns�ri�ed ��NA. The ri�oso�e st���s �t the
�NA:�NA jun�tion. �uro�y�in then �inds to the ri�oso��� A site. The n�s�ent po�ypeptide
is there�y tr�nsferred to puro�y�in. The resu�ting �o���ent�y �inked �o�p�ex ��n �e used
for se�e�tion experi�ents. �eprinted fro�: S�h�ffitze� C et ���� J I��uno� Meth 231:119-135;
©1999 with per�ission fro� ��se�ier. 90
with prokaryotic coupled transcription and translation (E. coli S30 extract) for selection of peptide
ligands 107‑109 and directed evolution of Taq DNA polymerase, 110 bacterial phosphotriesterase 111 and
DNA methyltransferase. 112 However, IVC has been used in conjunction with coupled transcrip‑
tion/translation in RRL to select active restriction enzymes from a randomized large (10 9 –10 10
molecules) mutant Fok I library. 103 Use of RRL in this way is made possible by a new inert emulsion
formulation that is compatible with coupled transcription/translation, 114 allowing an expanded
range of vertebrate protein targets that may be difficult to express as soluble and functional in S30
(bacterial) or wheat germ lysates.
Screening
In Vitro Expression Cloning (IVEC)
IVEC 115,116 is another approach that uses RRL for coupled transcription/translation to identify
genes and elucidate protein interactions with other molecules. Using this method cDNAs or small
plasmid pools (50–100 clones) are expressed and then assayed for a specific function. Plasmids from
the positive pools are further deconvoluted and rescreened (Fig. 4). The process is repeated until a
single positive plasmid has been identified. IVEC is only successful if the specific function assayed
can be distinguished from the endogenous activity in cell‑free RRL. IVEC has been successfully
used to identify protein substrates, 117‑120 protein‑protein interactions, 121,122 enzymatic activity, 123‑125
protein‑DNA interactions, 126 and phospholipid‑protein interactions. 136
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