Biology Reference
In-Depth Information
construction, protein expression, and functional analysis of the expressed protein.
Unfortunately, this process is time-consuming and costly (both in terms of funds and
human capital), since it is primarily a serial process where only a single or a few proteins
are produced simultaneously. High-throughput production of proteins using CFPS systems
is now addressing this challenge. Valuable for studying the synthesis of many proteins
simultaneously, protein microarrays are being developed at a rapid pace for improved large-
scale protein expression and purification.
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The basic principle of protein microarrays is that
DNA encoding a protein target of interest is printed onto a glass slide in a physically
isolated location, then CFPS synthesizes the target of interest.
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To improve protein
isolation and stability, proteins are commonly engineered to include epitope tags (e.g.
C-terminal glutathione
S
-transferase tags) which bind the protein products to antibodies
immobilized on the slides.
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In one exemplary report, parallel large-scale expression of
more than 13 000 human genes using a wheat germ extract has been achieved.
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Because
the cell-free approach obviates the need to synthesize, purify, and immobilize proteins
separately, it allows for proteins with novel characteristics to be quickly generated and
analyzed. Further efforts to integrate gene synthesis on chips for protein analysis promise
even greater capability.
CFPS offers a rich and versatile platform for protein engineering. Specifically, the cell-free
system enables direct control of the environment to efficiently drive protein evolution to
contain the desired traits.
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Two major protein evolution strategies are in vitro
compartmentalization and ribosome display. In vitro compartmentalization (IVC) involves
the generation of an emulsion to create cell-like compartments.
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Ideally, each
compartment contains a single gene and all reaction components necessary for protein
transcription/translation. These separate compartments trap all gene products within an
individual compartment, preventing cross-contamination. The reaction components may
either be present all at once or added in several phases.
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The formation of compartments
down to the femtoliter scale has made IVC by emulsion particularly attractive for
high-throughput protein evolution (
Fig. 15.3
).
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In contrast, the low-tech physical method
of IVC by microtiter plate has high reagent costs and the screening of one million genes could
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FIGURE 15.3
In vitro compartmentalization by emulsion for protein evolution: (12) Cell-free transcription/translation machinery and
mineral oil form water in oil emulsions. (3) In each emulsion, a single gene is transcribed and translated. (4) Proteingene
linkage may be performed prior to selection. (5) Proteins are selected based on binding, catalytic, or regulatory capability.
(6) Alternately, proteins may be selected through a fluorescence-assisted droplet sorting system (FADS). (7) The genes
for selected proteins undergo a new cycle of IVC.
