Biology Reference
In-Depth Information
cost over $2.4 million.
104
The implementation of new ultrahigh-throughput microfluidic
systems have dramatically reduced these costs.
105
In one such system, the use of picoliter-
sized droplets, low polydispersity emulsions, and effective fluorescence sorting allowed the
screening of one million genes in less than an hour at a cost of less than $31.
104
As a complement to IVC, ribosome display provides a simple mechanism for evolution of
proteins that will bind to a specific ligand.
106,107
The ribosome display process begins with
a DNA sequence or library of sequences containing a spacer sequence lacking a stop codon.
Due to the spacer sequence, the ribosome remains bound to the mRNA
protein complex
post-translation and the nascent polypeptide is able to successfully fold.
101
The mRNA
protein
ribosome complex can then be exposed to a surface-immobilized ligand for
binding. Weak-binding complexes are washed away, leaving only high-affinity complexes.
The mRNA of the highest affinity complexes are recovered, reverse transcribed, PCR
amplified, and then collected for future selection rounds.
107
In one example, ribosome
display enabled the rapid selection of a designed ankyrin repeat protein which binds the
cancer-relevant epidermal growth factor 2 (Her2) at high selectivity and nanomolar
affinity.
108
Beyond protein microarrays and protein evolution, the synthesis of synthetic proteins is
another frontier application of CFPS. While Hecht and coworkers have elegantly shown the
ability to synthesize de novo proteins with unique functionality from combinatorial
libraries,
109
we focus our discussion here on efforts to expand the chemistry of life by the
introduction of unnatural amino acids. Efforts to use CFPS for unnatural amino acid
incorporation are beginning to grow. This is because of recent advances enabling cost-
effective, high-level CFPS systems, and advantages over in vivo approaches. Namely, there
are no transport issues for unnatural amino acids, and there is greater flexibility for
reprogramming the translation system.
110
288
The incorporation of unnatural amino acids to create novel proteins has been performed by
globally replacing a natural amino acid with an unnatural analogue, or by site-specifically
incorporating the unnatural amino acid while maintaining the natural amino acid cannon.
The global replacement method can be performed in the cell-free system by simply not
adding an amino acid such as methionine, and adding an unnatural amino analogue in its
place. In a recent report, methionine was globally replaced with azidohomoalanine to
enable the efficient attachment of proteins to virus-like particles for the development of a
B-cell lymphoma vaccine.
31
While simple in implementation, the global replacement
method is more likely to adversely affect protein function due to the loss of methionine
from the amino acid canon.
111
A number of site-specific incorporation methods have been developed. However, one of the
highest yielding and transferable methods involves utilizing an orthogonal tRNA/tRNA
synthetase pair specific to the amber stop codon, as developed by Schultz and coworkers
.
112
The expression or addition of the tRNA/tRNA synthetase pair is relatively straightforward in
both in vivo and cell-free systems. However, due to the insoluble nature of some unnatural
amino acids and transport limitations of the unnatural amino acid into the cell, the efficient
incorporation of some unnatural amino acids in vivo can be challenging.
29
Because of the
direct access provided by cell-free systems, these limitations can be overcome.
29,30,111
By
optimizing the tRNA/tRNA synthetase pair and using a continuous exchange cell-free system
with linear templates, Ozawa et al. recently produced protein yields of over 2 mg/ml for a
protein with a single unnatural amino acid mutation.
113
For site-specific incorporation, the presence of release factor 1 (RF1) in cell-free extracts can
compete with the exogenous tRNA for the amber stop codon.
114
A significant improvement
in site-specific multisite incorporation of unnatural amino acid protein yields has been
made by producing an RF1 knockout strain of
E. coli
. With this strain, protein yields reached
