Biology Reference
In-Depth Information
times and was much more complicated than 2D origami.
58
New technology by the Yan
group has created a more sophisticated version of DNA origami termed DNA kirigami that
involves folding and cutting DNA into topological objects.
59
Additionally, the same group
created a strategy to engineer 3D DNA structures with complex curvature.
60
As the
fundamental design principles of complicated architectures become more and more
elucidated, including computer-aided design caDNAno,
61
this field is now turning to
applications.
1
Key among these is the creation of DNA nanochips that can be used to
observe single molecule behavior of DNA-binding enzymes,
62
and DNA nanorobots for
medical therapeutics and medical diagnostics.
63
In particular, the recent demonstration that
DNA nanorobots can target cancer cells and deliver an antibody payload
63
is expected to
usher in a new era of DNA device utilization.
Proteins
In contrast to in vitro efforts in DNA and RNA that mainly center on building and
understanding nucleic acid circuitry, major in vitro efforts in proteins have been focused on
synthesis and evolution strategies. Indeed, a technological renaissance has reinvigorated cell-
free protein synthesis (CFPS) technologies over the past decade.
4
This progress has realized
protein yields exceeding grams of protein produced per liter reaction volume, cost
reductions of multiple orders of magnitude, and microscale to manufacturing scale
production. Here we discuss both bottom-up and top-down approaches to CFPS, along with
frontier applications enabled by recent advances.
BOTTOM-UP SYSTEMS
The bottom-up approach to CFPS centers on using purified components. Pioneered by Ueda
and colleagues, the most prevalent bottom-up system is the PURE system.
3,64
In the PURE
system, cellular machinery necessary for translation is independently overexpressed,
purified, and combined in a test tube.
65
An advantage of the PURE system is that the
researcher is afforded the greatest management of every aspect of the protein synthesis
process. Indeed, the ability to mix and match nearly any component of translation has
proven remarkably useful for efforts to fold proteins
64
and to incorporate nonnatural amino
acids.
28
The main disadvantage to this system is its cost. The necessity of expressing,
purifying, and adding each component greatly increases the reagent cost and time required
compared to top-down systems.
66
A cost comparison of the CFPS systems discussed herein
is provided as
Table 15.1
.
284
TOP-DOWN SYSTEMS
The top-down approach involves the modification and engineering of crude cell extract for
protein production. Although any organism can potentially provide a source of crude
lysate, the most frequently utilized crude extract systems are made from
Escherichia coli
,
wheat germ, rabbit reticulocytes, and insect cells.
4
The
E. coli
-based CFPS systems are currently the most widely used due to lower labor
requirements for cell growth and extract preparation, lower-cost energy sources, higher
yields, greater rate of protein synthesis, and commercial scalability.
2,67
Given the significant
number of proteins previously expressed in this system and its high linear scalability (from
15
L to 100 liters), the
E. coli
-based CFPS currently demonstrates the greatest versatility.
4
However, this cell-free system still faces post-translational modification challenges, although
a new report has described glycosylation in these systems for the first time.
68
Other bacteria-
based cell-free systems have been developed, notably from thermophiles such as
Thermus
thermophilus
.
69
μ
Eukaryote-based CFPS systems are mainly derived from wheat germ, rabbit reticulocytes,
and insect cells. Wheat germ extract-based cell-free systems provide the highest yields of the
