322 15 Cell‐Free Protein Synthesis
and (iii) can lack robustness and predictability due to several reasons: the com-
plexity, the host-dependent gene expression and protein folding/function, the
necessity of product export from the cell membrane for improved production,
and the toxicity of high levels of expressed proteins to the host. CFPS can address
many of these limitations to help complement existing technologies, but there
are remaining immediate challenges. For example, the field is limited by its abil-
ity to produce posttranslationally modified proteins at high titers, particularly
those with human patterns. Moreover, we still do not have the protein equivalent
of PCR. Further, inefficiencies in site-specific incorporation of ncAAs limits
innovation. By addressing these challenges, we anticipate that cell-free systems
will continue to penetrate and be recognized for value by industry. Given the
capability to modify and control cell-free systems, CFPS holds promise to be a
powerful tool for systems biology, for synthetic biology, and as a protein produc-
tion technology in years to come.Definitions
Cell-free protein synthesis is the process of translating proteins in lysates
In vitro is the processes performed outside of their biological context, e.g. pro-
tein synthesis occurring outside the cell
Noncanonical amino acid is any amino acid outside the 20 canonical amino
acids
Glycosylation is the addition of sugar moieties to proteins
Antibody is the protein of the immune system that recognizes and neutralizes
pathogens
Membrane protein is the protein that is associated with or integrated into a
cellular membrane
High-throughput is the capability of being performed many times in parallel
Protein screening is the process of testing one or more proteins or protein variants
in one or more contexts to determine properties of the protein(s) or optimize
Genetic circuit is the engineered use of DNA sequences to control biological
reactions and programsAcknowledgments
The authors thank C. Eric Hodgman for his advice and discussions regarding the
manuscript. MCJ gratefully acknowledges funding from the National Science
Foundation (Grant Number MCB-0943393, Grant Number DMR-1108350, Grant
Number MCB-1716766), the Air Force Research laboratory (FA8650-15-2-5518),
the Army Research Office (W911NF-11-1-0445 and W911NF-16-1-0372), the
Human Frontiers Science Program (Grant Number RGP0015/2017), the Office of
Naval Research (Grant Number N00014-11-1-0363), the DARPA Living Foundries
Program (N66001-12-C-4211), the DARPA Biomedicines on Demand Program
(N66001-13-C-4024), the Defense Threat Reduction Agency (GRANT11631647),
the Department of Energy (Grant Number DE-SC0018249), the David and Lucile