Biology Reference
In-Depth Information
ADVANTAGES ACHIEVED BY DIRECTLY INFLUENCING REACTION CONDITIONS
As mentioned above, cell-free biology provides the ability to directly manipulate and
modulate reaction conditions. We now highlight several illustrative examples where ionic
strength, pH, redox potential, hydrophobicity, and translation components were tuned. The
overall ionic strength and the relative concentration of a specific ion can significantly impact
the structure and activity of many enzymes and proteins.
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For example, macromolecular
protein structures such as virus-like particles have been shown to assemble at higher
efficiencies as the ionic strength is increased.
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Viral RNA polymerases are commonly
inhibited by high concentrations of zinc.
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Cell-free biology enables direct optimization of
the ionic strengths for both the performance of the enzyme machinery and the activity of the
product protein. By utilizing cell-free translation and assembly reactions, the highly efficient
translation and assembly of macromolecular virus capsids has been demonstrated.
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Also, the cell-free assembly of the human hepatitis B virus and human papillomavirus capsids
were observed to be the highest at ionic strengths that would be cytotoxic to most cells.
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Cell-free biotechnology also enables the ability to directly control the redox potential, which
is not possible in cells. In an exemplary illustration, Zawada et al. carried out a
combinatorial optimization of reaction conditions for both protein expression and folding.
2
By modulating the redox potential and disulfide bond isomerase concentrations, they
achieved greater than 95% solubility for a protein containing multiple disulfide bonds. This
is in contrast to in vivo studies where correctly folding such complex proteins is very
difficult. Indeed, cell-free production of many active disulfide-bonded proteins and
disulfide-bonded macromolecular complexes has been achieved, including
E. coli
alkaline
phosphatase, human granulocyte-macrophage colony-stimulating factor,
Candida antarctica
lipase B, human lysozyme,
Gaussia princeps
luciferase, and the Q
virus-like particle which
contains up to 180 disulfide bonds.
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Although a subtle point, the less-crowded
environment of the cell-free reaction (relative to in vivo concentrations) helps provide for
improved protein folding.
β
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The open nature of cell-free translation also enables the use of several synthetic approaches
to increase solubility of highly insoluble proteins such as membrane proteins.
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In many
instances, detergents have been used to alleviate protein solubility issues.
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In one example,
detergents and chaperone proteins were successfully used in the cell-free system to produce
an active form of an insoluble cancer-linked protein that had never been produced
in vivo.
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More recently, others have focused on the use of nondetergent surfactants,
amphipols, and fluorinated surfactants to increase solubility without damaging protein
structural integrity.
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Other recent breakthroughs have included insertion of insoluble
proteins into synthetic liposomes with verified postinsertion functionality.
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Recently, advances in protein-producing scaffold technology have produced synthetic
hydrogels capable of cell-free production yields approaching those of any solution-based
cell-free systems.
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Hydrogels are scaffolds made up of linked networks of strongly
hydrophilic polymers and mimic hydrophilic physiological environments.
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These hydrogels
have been reported to be compatible with various cell-free systems, and capable of
generating protein yields up to 5 mg/ml.
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APPLICATIONS
Improvements in productivity, scale, and complexity of recombinant protein synthesized
have rapidly expanded the utility, and now industrialization, of CFPS systems. In this
section, we highlight several emerging applications, focusing on protein microarrays, protein
evolution, and synthetic proteins.
Currently, the synthesis of proteins for functional analysis, structural genomics projects, or
the identification of novel characteristics is a multi-step task. These tasks involve gene/vector
