History and evolution of cell-free protein synthesizing systems
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Acrobat) The very earliest studies of protein synthesis in cell-free systems included those
of Siekevitz and Palade in the early 1950's. Palade's delightful account of how the
foundations of cell biology were built is indispensable reading for anyone entering the
field. The first of the modern cell-free protein synthesizing systems (circa 1970)
evolved from the "readout" systems such as used by Redman et al. in the late 1960's.
In these "zoo" systems purified large and native small ribosomal subunits (including
bound initiation factors) were combined with "pH 5 enzymes" (aminoacyl tRNA
synthetases and other factors) and energy generating systems, etc. WG (developed in
the early 1970's) represented an advance in two ways over the zoo system. First, it
was a single extract that contained all desired components, therefore much easier to
prepare. It was also much more active in terms of both total synthesis, synthesis over
background and rounds of translation per molecule of mRNA. Its disadvantage was
that it gave a high proportion of "early quitters" and "false initiators" which appeared as
a ladder of smaller bands down the gel. As a consequence of this it was a system good
for only synthesizing relatively small proteins. Shortly thereafter the RRL system was
popularized. The advantage of this system was that it was even more powerful than
WG in terms of rounds of synthesis and in making full-length proteins. Two key
obstacles were overcome in the early 70's that made it all the more attractive. The
work of Hunt and others elucidated the hemin inhibited cascade of translational
regulation which otherwise rapidly destroyed the activity of the lysates. Secondly, the
introduction of micrococcal nuclease allowed endogenous mRNA (i.e. mainly globin
mRNA) to be removed. Another problem with RRL remains to this day: the
endogenous cold globin concentration is enormous (in the 100 mg/ml range). This
limits the amount of total product you can analyze directly on a gel, makes sample
preparation a headache (you need to use high concentrations of DTT to get complete
reduction; TCA precipitation of undiluted lysate generates a precipitate with the key
properties of cement), distorts both the gel and banding pattern, etc. An unanticipated
advantage of RRL proved to be that protein translocation across the ER membrane
proceeded with higher efficiency than in WG. In the meantime, the introduction of an
organic solvent "flotation" step in the preparation of WG extract allowed the separation
of "good" from "bad" embryos, presumably on the basis of their water content, that is
those that were truly dormant embryos would float in a cyclohexane/carbon
tetrachloride cocktail. These floated and dried embryos were found to make an
extremely active protein synthesizing extract. Together with the newly introduced
RNAse inhibitor from human placenta (RNAsin) this procedure placed the WG system
on at least even footing with RRL for expression of high mw products with efficiency.
Thus today, both systems have their place and value and their limitations. It is
important to know these in order to make a rational choice of which system to use.
References: Erickson AH & Blobel G. (1983) Meth Enz 96:39-50
