Ministry of Higher Education and Scientific Research

Duhok Polytechnic University

Shekhan Technical College of Health
Higher Education Committee

Fatima Esmael Hassan

Supervised by

Dr. Meqdad Saleh Ahmed

(2023 - 2024)
Table of Contents
1. Introduction.............................................................................................. 1

2. Fundamental Principles ........................................................................... 2

2.1. Transcription ...................................................................................... 5

2.2. Translation ......................................................................................... 7

3. Applications ........................................................................................... 10

3.1. Protein Synthesis ............................................................................. 11

3.2. Genetic Engineering ........................................................................ 12

3.3. Drug Discovery ............................................................................... 13

4. Advantages ............................................................................................ 14

4.1. Time Efficiency ............................................................................... 15

4.2. Cost Effectiveness ........................................................................... 16

4.3. Flexibility......................................................................................... 18

5. Limitations ............................................................................................. 19

5.1. Protein Folding ................................................................................ 20

5.2. Post-Translational Modifications .................................................... 21

6. Conclusion ............................................................................................. 21

7. References.............................................................................................. 22
1. Introduction
In this chapter, we introduce the concept of cell-free gene expression (cell-
free systems, cell-free protein synthesis, and cell-free transcription-
translation), review the history, and demonstrate the significance across
numerous scientific fields. This section also includes a list of cell-free
systems and application examples. In the next section, we provide reviews of
the necessary elements or parts of the cell-free systems, such as genetic
components (expression vectors, regulatory DNA, coding DNA, reporter
DNA, control DNA), expression vectors, regulatory DNA, coding DNA,
reporter DNA, and control DNA. Throughout this review, we aim to
highlight how the development of the elements or parts has driven the
enhancement of in vitro reaction processes. (Garenne et al.2021)

Cell-free gene expression systems are platforms for efficient and rapid
synthesis of protein and RNA molecules in a test tube. The basic ingredients
of the systems are several compartments and building blocks (DNA, RNA,
amino acids, salt, cofactors, an energy source molecule). Because of their
modularity, robustness, and flexibility, cell-free systems have strengths over
whole-cell systems and offer the rapid screening of a gene circuit and the
optimization of a synthetic protein. The systems are applicable across
numerous fields. Since the first cell-free system, in vitro (cell-free) protein
synthesis (cf-ivps), most developed and the most popular cell-free gene
expression systems utilize bacterial proteins, such as T7 RNA polymerase,
ribosome, and its associated factors. (Silverman et al., 2020)

1
2. Fundamental Principles
Living cells (in vivo conditions) are mostly closed and dynamic systems that
combine a countless series of chemical reactions, namely metabolism, to
maintain life. The essential aliveness of the cell is difficult to replicate. The
first cell-free translations were conducted by Harline and Nirenberg in 1961.
The adaptive potential of living organisms is very high, and it is largely
based on the synthesis of particular products present in very low amounts.
The generation of greater diversity is a precursor to the process of finding
molecular recognition elements in nature and the process of achieving
optimal molecular recognition, monovalence, or low polyvalence. Cell-free
studies are a necessary staging of the generation of diversity and inquiry of
affinities. (Sigalova et al.2021)

Figure 1: Equipment needed for CFE

2
Figure 1 - a | Prepare: Gather disposable gloves, face masks, safety glasses,
calibrated pipettes, tube racks, a vortexer (gentle vortexing <4,000 rpm for
<10 s), a minifuge, and 70% ethanol for sterilization. b | Set up a custom
CFE system with six aqueous solutions (DNA template, lysate, energy mix,
amino acid mix, cofactors, magnesium) in 1.5-ml tubes or microwell plates.
c | For time course fluorescence, program the plate reader, assemble the
reaction at desired temperature, and monitor in real-time. For endpoint
fluorescence, incubate in an incubator, analyze data. For non-fluorescent
products, use SDS-PAGE.

3
4
Figure 2: Overview of a typical CFE workflow

The chapter starts with a comparison between in vivo and in vitro protein
synthesis, and it develops the idea of cell-free gene expression as a selective
alternative to whole-cell systems. Following is an overview description of
the mechanism of protein synthesis, with a focus on the most important
modules for cell-free expression. The last part of the chapter presents some
chemically defined systems that include a number of biocomponents of the
protein synthesis machinery. To calibrate the reader's background, this
chapter opens with a brief contrast of in vivo and in vitro protein synthesis
and discusses the applicability of cell-free systems to directed evolution.
Although the emphasis is on systems that perform a portion of the protein
synthesis machinery, finally, we introduce systems that incorporate a
significant number of actual biocomponents. (Silverman et al., 2020)
(Zawada et al.2022)

2.1. Transcription
At the one extreme end, the free ribonucleotides can be seen as the first
reactants, then the monophosphate nucleotides, then pyrophosphate, and as
the last, the holopolymer is released. Depending on the secondary structure
of the template and the nature of the polymerase molecule, the
concentrations of the accumulating main intermediates change. In general,
two kinds of intermediates can accumulate in the reaction progress: RNA
molecules with partially synthesized sequences and DNA-RNA hybrid
duplexes on the DNA template. If ssDNA templates are used, the main
intermediates are the corresponding RNA molecules. If the template has
long flanking dsDNA regions, the main reaction intermediates are the
stationary dsDNA-RNA hybrids. In the latter situation, the ssRNA is

5
released after the duplex translocation to the polymerase molecule. After the
RNA transcript is displaced from the polymerase, the elongation of the RNA
molecule can resume. (Garenne et al.2021)

One of the first steps in a cell-free gene expression process is declaring the
DNA. In the most popular cell-free gene expression system, Escherichia
coli, the major components such as E. coli or T7 RNA polymerases and NTP
are also added into the base reaction. The main substrate for both
polymerases is DNA, and the dependency of the reaction rates on the added
DNA concentration forms a popular Michaelis-Menten-like hyperbola. In
contrast to the classical Michaelis-Menten enzymology models, the
polymerase-driven transcription of the DNA molecule can result in the
elongation of the entire DNA or RNA transcript. As a result, the cell-free
reaction can undergo decay at high DNA template concentrations in the
reactions containing various efficient RNAse molecules. (McNally et
al.2023)

6
Figure 3: Key steps for Escherichia coli lysate preparation.

This figure shows the Key steps for Escherichia coli lysate preparation. The
Process typically requires 3 days and involves four steps. a | Cell growth to
OD600 of 2–4 in 2.5-l baffled flask and collection by centrifugation. b |
Washing cells three times in an iso-osmotic buffer at 4 °C, and cell lysis
performed at 4 °C using the same iso-osmotic buffer. c | Incubation of the
lysate in 14-ml culture tubes at 37 °C in a shaker incubator for 2 h. d |
Dialysis of the lysate at 4 °C in a 2-l beaker with a magnetic bar to stir the
buffer, and storage at –80 °C. The dialysis step is optional, providing a lysate
with greater efficacy for cell-free gene expression (CFE).

2.2. Translation
Recently, more effort has been directed toward understanding, and often
simplifying, cell-free translation. However, evaluating trends in this field is

7
complicated by differences in techniques and components, and by the more
recent lack of detailed technical reports. Increasingly, highly regarded
researchers favor crude lysates over reconstituted systems, and while
interpretation could be biased because crude lysates are simply a more useful
tool for charting novel discoveries, it is more likely that the advantage of a
more accurate representation of the in vivo situation is recognized. Lund et
al. examined the changes in mRNA translation in three in vitro systems as
compared to the in vivo situation in both bacterial and eukaryotic cells. They
found that bacterial translation in T7 S30 and E. coli lysate compared well,
while rabbit reticulocyte lysate and wheat germ extract poorly represented
mammalian translation. This result is not surprising, and the generality of
this statement is supported by others. What is less clear is why in vitro
systems should underrepresent translation of a particular system more poorly
than others; in an unrelated study, Flint et al. also reported that wheat germ
extract was the best S30 system tested. In contrast, Beck and Pohl’s work
with eukaryotic transcriptionally active extracts agrees more closely with the
findings in the bacterial systems. (Sharma et al.2023)

In addition to a transcriptionally active extract, a cell-free system must also

contain everything required to translate the message produced. Originally,
crude cellular lysates were used for cell-free translation, which ultimately
led to the elucidation of the basic mechanisms of the process. These ceased
to be the predominant source after lysates were fractionated and components
such as ribosomes and the necessary ligands could easily be separated.
Purified bacterial and eukaryotic ribosomes, tRNAs, and initiation,
elongation, and release factors were reconstituted into functioning cell-free
systems in the lab. (Cui et al., 2022)

8
Figure 4:

Figure 3 shows Monitoring transcription and translation dynamics in cell-

free expression. (A) Construct of the pEXP5-NT/6xHis mCherry F30-
2xdBroccoli plasmid containing a constitutive T7 RNAP-mediated promoter
expressing 6xHis mCherry with an F30-2xdBroccoli RNA aptamer tag. The
small-molecule dye DFHBI becomes fluorescent upon binding with the
Broccoli RNA aptamer. (B) mRNA and mCherry protein expression levels
over time from bulk PURExpress CFES titrated with varying concentrations
of pEXP5-NT/6xHis mCherry F30-2xdBroccoli DNA plasmid. (C) mRNA
and mCherry protein expression levels over time from bulk PURExpress
CFESs titrated with varying concentrations of purified 6xHis mCherry F30-
2xdBroccoli RNA transcripts. Solid lines and shaded areas correspond to
mean and standard deviation values from triplicate experiments,
respectively. Dashed lines are resource-limited CFES model fits. (D)
Illustration of the resource-limited gene expression model for CFESs.
Parameters are kr: RNA transcription rate, Kr: dissociation constant between
RNAP and DNA, δr: RNA degradation rate, kp: protein translation rate, Kp:
dissociation constant between ribosome and RNA, kmat: mCherry maturation
9
rate, δTsR: TsR degradation rate, δTlR: TlR degradation rate, Kl: Michaelis–
Menten constant for TlR degradation, a: scaling factor for consumption of
TsR with transcription, b: scaling factor for consumption of TlR with
translation, and τd: time delay for protein translation.

3. Applications
Although perhaps of more theoretical interest, the examples mentioned in
the preceding paragraph might be obtained more rapidly from CFGE than by
cloning and large-scale expression. Researchers should take into account
that CFGE does not always provide the highest protein yields even if it can
provide protein rapidly and in large amounts. Other factors might mitigate
against CFGE, making cloning and expression of proteins in an E. coli or
other production strain the proper choice to obtain the desired amount of
protein with the required properties. Finally, CFGE is complimentary to
other methods used to express proteins; there is not an exclusive use for
CFGE in any particular application nor in all research areas that would
benefit from efficient expression. (Peťková et al.2021)(Silverman, 2021)

Applications of CFGE are quite broad as shown by the more than 20 clinical
research, interventional and case reports that have been reported. As a
research tool, the largest single application of CFGE has been in the area of
proteins used therapeutically or as reagents. This includes antibody
fragments, hormones such as insulin or erythropoietin, blood clotting
factors, enzymes, and small secreted proteins. Other applications involve
proteases, protein scaffolds, and proteins with industrial uses such as
cellulases for ethanol production, asparaginases for reducing toxic
concentrations of asparagines in patients, and antigens for generating

10
antibodies. Proteins to be used in CFGE are typically encoded with AgNuc
and flanked with ribosome binding sites appropriate for in vitro translation;
lysis of the producer cells would release large amounts of the protein into the
surrounding medium. (Qiao et al.2020)

3.1. Protein Synthesis

The first cell-free protein synthesis of an active enzyme was demonstrated
by Nirenberg and Matthaei in a slightly different in vitro system in which
ribosomes were "programmed" by RNA. They were able to reconstitute the
genetic code by selectively introducing triplet RNA sequences into protein
polymers, using 20 natural amino acids and a synthetic RNA polymer. This
experiment showed that an RNA sequence could instruct the incorporation
of defined amino acids into a protein polymer, establishing the connection
between the nucleotide sequence and the protein sequence, and confirming
that mRNA is the template for protein expression in the cell. More recently,
research has been focused on the rational engineering of the cell-free
machinery to achieve efficient recombinant protein synthesis, as well as the
synthesis of non-standard natural and unnatural proteins by varying the
substrates supplied in the system. (Liu et al.2020)

Cell-free protein synthesis (CFPS) is the process of protein synthesis in a

cell-free system, which is a way to produce a large amount of protein using
the biological components of living cells without the cells themselves. Cell-
free protein synthesis is performed by mixing the extracted cellular
machinery for transcription and translation with the necessary substrates
(usually nucleotides, amino acids, energy sources, and buffering salts). The
great advantage of cell-free protein synthesis, like in vitro transcription, is
the possibility of fine-tuning and optimization of the reaction conditions.

11
Additionally, CFPS enables the synthesis of membrane proteins and other
proteins that are typically difficult to overexpress when using in vivo
recombinant expression. In various cell-free systems, demonstrated the
synthesis of several important human proteins, such as insulin, successfully,
providing a first glance of the biomedical applications of SoloPURE. (Chiba
et al.2021)

3.2. Genetic Engineering

Building on the theory that the network is a biomolecular switch, a 2005
computational circadian clock model assumed a highly non-linear p53
dynamics. Because cell-free systems of gene expression are making it
possible to examine network components and their relationships, templates
for the p53 and mdm2 genes are added to the reaction mix. When ATP,
GTP, UTP, and CTP are introduced, the genes expressed in cell-free systems
are translated to produce either p53 or Mdm2. Meanwhile, the reaction mix
has been made as competent as possible to support cell division. Protein
synthesis is upregulated, and protein levels are quantified. In the presence of
cisplatin, a chemotherapy drug that causes DNA damage, p53 amounts rise
dramatically in about 1 h. In fact, the dynamics reproduce the in vivo p53
response. (Lafata et al.2021)

Life scientists have identified myriad genes that are expressed in response to
extracellular signals to regulate cellular circuitry. However, cellular
complexity poses an obstacle, hindering investigation of how networks
propagate and integrate signal responses derived from these cascades.
Consequently, researchers are turning to cell-free gene expression systems
as platforms to probe interactions within and between regulatory circuits.
Because such systems are stripped to their core reactions, signals can be

12
more easily introduced and events that lead to network responses quantified.
The p53-Mdm2 system is composed of a signal transduction network that
involves a conserved family of proteins. The p53 master regulator protein is
activated by DNA damage, then binds to p53 response elements that regulate
expression of target genes. Transcription of mdm2 by p53 starts an
autoregulatory process. The amount of Mdm2 protein influences the p53
protein amount. Both proteins interact with other proteins to produce p53
sufficiency or deficiency along with the ratio of Mdm2 to p53 protein levels
- vital regulatory components to maintain homeostasis, stability, and wild-
type cells. (Silverman et al., 2020)

3.3. Drug Discovery

Cell-free gene expression screening identifies successful sequences that live
cells reject. Following the cell-free screen, only the identified lead sequences
are tested further. Kits are available for many levels of downstream analysis.
What do you do with your synthetic DNA once it's made? Where can PCR
and Gibson Assembly lead you next? Certainly starting cell-free synthesis of
your optogenetic system overnight is a great beginning. Other platforms
have a similar philosophy to simply serve the non-biologist for
incompatibilities. Kits are available globally and there are numerous third-
party reagent sales. Do not be fooled by companies national. They do not tell
the full story. Companies specialize in developing certain areas of DNA
synthesis. (Galiñanes Reyes, 2021)

Two other areas stand out in the potential application of cell-free gene
expression to address specific challenges. First, cell-free gene expression
offers a way to experiment with genetic variations without large-scale
robotic screening. Traditional robotic drug discovery requires hundreds of

13
thousands to millions of plates. While it is certainly possible to screen such
large volumes over several months or years, doing so takes time and is
expensive. Cell-free gene expression can be used to test an entire library of
genetic variations on a desired target in a single afternoon. Putting cell-free
gene synthesis together with a cell-free gene expression platform allows
researchers to broaden their research and increase success without
overwhelming their collaborators in high-throughput screening. (Abdueva et
al., 2024)

4. Advantages
In vitro protein synthesis systems underpin a wide range of uses in
biotechnology and as a platform for bio-based manufacturing, including
applications either using the gene expression system directly for in vitro
protein production, or indirectly, for example, for chemical synthesis,
sensors, display technologies, diagnostics, and medical applications. In
many of these applications, cell-free lysate production platforms like the
ones described in Section 4 eventually provide the enzymes, RNA
polymerases, cell membrane components, ribosomes, transfer RNAs, and
more to culminate into an active gene expression system. Of these system
constituents, the most defining part unique to cell-free expression is the
isolation of cellular compounds such as the gene expression apparatus or
production platform that can be used with lysate in an application at hand.
(Brookwell et al., 2021)

Cell-free gene expression (CFE) offers some advantages over conventional

in vivo production methods. These advantages can be utilized for
biotechnological applications. The most prominent benefit of in vitro protein
synthesis is the rapid cell-free gene expression. Other highlights of cell-free
14
expression systems are their ability to utilize substrates and express products
not easily tolerated by living cells, such as RNAs and highly toxic proteins.
In addition, in vitro methods produce less waste compared with processes
relying on whole cells, and output scales linearly within the limits of
enzymes, substrates, energy, and cofactors available in the reaction.
(Garenne et al.2021)

4.1. Time Efficiency

After some rounds of optimization, in-house expression may still take at
least 24 hours after transcription. Despite that, cloning and pre-initiation will
take up to 3 days. This is the time we take to synthesize a gene from scratch.
Therefore, adding another 24 hours of gene expression reaction will take a
total of 4 days. Conversely, tasks such as gene PCR, post-PCR purification,
Gibson Assembly, colony PCR, sequence analysis and in-house DNA
recombinant assembly PCR may also take up to 3 days, or even 4 days,
depending on how many gene copies were amplified. Subsequently, deep
sequencing commercial services will always take an additional 1-2 weeks to
be delivered. In some cases, time estimation is not enough. For example, it
may take more time to clone a short gene fragment for in-house expression
than what it takes to synthesize the complete coding region of a gene flanked
by the necessary DNA sequences for in general less than 48 hours. (Cheng et
al., 2023)

Time is a very important factor in daily gene expression laboratory

procedures. For example, cloning can take around 3-7 hours per gene,
performed by skilled personnel and if done in parallel. It will also require 4-
15 days of active reaction time, depending on the wet lab infrastructure. In
the case of E. coli, it takes approximately 3 hours to grow a 50 mL TB

15
culture starting from a single colony up to the mid-log phase. Cell lysis is a
fast step and can be done in approximately 15-20 minutes and pre-initiation
can also easily take approximately 1.5 hours, if not more. Energy
regeneration can take another 2 hours to be completed after the addition of
the complete membrane fraction plus the ion carrier. Alternatively, if the
energy regeneration solution is added after the protein synthesis cocktail,
this process will also take 10 hours. Whereas the transcription/RNA
synthesis step will require between 2 to 4 hours to be completed. The
following steps can be optimized too: polystyrene binding, T7 RNA
synthesis, among others. Later steps such as protein degradation and folding
will also take several hours. (Wang et al.2022)

4.2. Cost Effectiveness

In silico design can also be shared, with groups drawing on each other's
expertise, beyond just sharing a batch of costly cell-free synthesis,
maximizing the sharing of scientific resources. The time savings for the
group that enables breakthrough sequences will be very large. Then, as more
reactors come online and are shared by the participants, the result of the
experiment will be not just an exercise in the stealthy exploitation of
government funding, but also a global reduction in infrastructure (and speed
increase) for cell-free translation of more and more complex protein
sequences, as well as a significant reduction in the cost of production only
partially being able to exploit the sharing of real space between the groups
and thereby, partially reducing the exploitation of government funds.
Because they can be directly engineered in the base extract, together with
infrastructure costs, the need for experts in cell-free protein synthesis
preparation should also decrease rapidly over time if file-based, high

16
complexity cell-free networks become widespread, an additional cost
savings that is not immediately obvious to proposal assessment committees.
(Wang et al.2023)

With the successful operation of complete systems today, scaled up to 1

litre, and estimated costs as low as under US$0.07 for a mere 100mg of
synthesized GFP, an important milestone has been achieved in the academic
field of cell-free protein synthesis for biosensors and membrane proteins.
The next significant demonstration that will advance cell-free systems into
the market is likely to be the scalability of whole genome synthesis. This is a
process used in various fields of synthetic biology, including cancer biology,
which is typically much more expensive when done in vivo. One potential
approach to financing a large-scale production endeavor in the current
research funding environment involves repurposing and sharing established
genetic synthesis facilities. Participants would bring their own sequences
that need to be synthesized in vivo, but with the use of certified fragments
that are ready for tunable, cell-free translation. After a rapid assembly of
species, the necessary wet-lab work would be performed, with a significant
portion of the shared costs going towards the in silico design phase.

The savings in cost that comes from being able to scale up cell-free protein
synthesis in a simple manner is one of the first advantages everyone notes
about the field. Being able to tune reaction conditions to a specific template
also means less protein is wasted. Major increases in throughput and
increased sample stability further decrease costs. By combining in vitro and
in vivo translation systems, the larger the yield needed, the less expensive
the per unit cost will be. Beyond simply reducing the cost of production of a
single template, openly sharing the cost of a single preparation of a batch of

17
extract among even just a few researchers can also make synthesizing
dozens of distinct proteins simultaneously significantly less costly than
traditional in vivo expression while also being less labor intensive. Due to
how scalable cell-free protein synthesis is, being able to exploit these
savings with automation is hence the most straightforward direction for the
future of the field. (Batista et al.2021)

4.3. Flexibility
With regard to the source of the synthesis elements and energy, CFPS can be
further divided into two types: extract-based and reconstitution-based.
Extract-based CFPS is generally cheaper, but shows reduced potential as the
source of contamination of the host cells, often limits the duration of
synthesis, and is less useful for the production of large proteins. By contrast,
reconstitution-based CFPS, although requiring commercially available
products for synthesis, is able to eliminate repressor masking and has
significant advantages of flexibility, robustness, and stability in many other
aspects. Among cellular synthesis elements, which are shown to affect the
efficiency of assembly in a CFPS, energy and ribosomal elongation
machinery that can accept orthogonal components are considered
particularly important, while cellular interactions of membrane proteins may
require cell envelope or proteoliposome. (Choi et al.2023)

Cell-free gene expression is a highly efficient technology of gene expression

that works in minutes in a test tube to produce proteins from any gene. The
CF protein synthesis technology is progressing and found applications in
large-scale protein synthesis, metabolic engineering, on-demand
biosynthesis, and cell-free synthetic biology. The primary advantage of cell-
free systems is that they operate without cell walls and cell division and can

18
express toxic proteins without limiting the growth and viability of the cells.
In choosing the appropriate cell-free system for the production of one
protein, the main strategies are to consider which purified components are at
which stage of synthesis and the compatibility with which synthesis
components of the tertiary structure of a protein. (Silverman et al., 2020)

5. Limitations
A main advantage of fed-batch methods over batch processes is that a
second, usually limiting, nutrient is added in the course of the reaction once
the first has started to be consumed. This allows for more cellular growth
and therefore increased reaction duration. When considering recombinant
production of proteins or even more complex macromolecules and
organisms, the addition of supplementary precursors not only influences the
duration of the reaction but can also be interesting when the stoichiometric
relationship between available substrates and expressed protein is
unfavorable. Batch substrate feeding by part of the cellular metabolism,
which becomes available through cellular lysis, has been thought to take
place in various E. coli and S. cerevisiae CFPS studies. However, real
substrate feeding is difficult to monitor in CFPS reactions, but more
importantly, it was recently shown that E. coli, as well as S. cerevisiae
extracts, consume ATP and precursor metabolites accumulated in the lysed
cell for CFPS of a model protein, implying that substrate feeding during
batch reactions is an artifact of cellular lysis and not of fed-batch
metabolism. Fed-batch methods pose particular challenges for CFPS
considering that the PCN will likely exceed maximal extract metabolic
capacity, leading to ATP and other precursor accumulation in the course of
the reaction. (Copeland et al.2021)

19
One of the main drawbacks in batch CFPS systems is the limited lifetime of
the reactions due to the rapid depletion of substrates and the accumulation of
inhibitory by-products. For this technology to be feasible at larger scales,
and particularly for applications in which high fluxes of production are
needed, it is necessary that the reactions last long enough to generate a
significant amount of product. Several strategies to extend reaction duration
have been employed and proposed, including glucose control or feeding, cell
extract treatment, metabolic regulation, and more recently oxygen
scavenging. (Chiba et al.2021)

5.1. Protein Folding

Proteins fold into a specific 3D structure via a stable energy minimum
governed by the amino acid sequence, known as the native state. Misfolded
states, called off-pathway or trapping local minima, can hinder this process.
Protein folding involves conformational changes, thermodynamics, and
geometrical constraints. Solvent-exposed residues collapse and interact to
form hydrophobic cavities. Charged moieties on protein surfaces recognize
each other through Coulombic forces. (Li et al.2020)(Sorokina et al.2022)

Cellular gene expression involves transcription and translation.

Transcription synthesizes messenger RNA (mRNA) with RNA polymerase.
Translation produces polypeptides based on the nucleotide sequence of
mRNA, with the help of ribosomes and transfer RNA (tRNA). tRNA's 3D
structure allows it to bring anticodons and the acceptor end close to each
other. The fold of tRNA is determined by weak atomic interactions. The
ribosome restricts tRNA movement, giving the rest of the structure a defined
shape. (Smolskaya et al.2020)

20
5.2. Post-Translational Modifications
Glycosylation is a crucial stage in protein formation, linking sugars to
proteins. It involves linking monosaccharides and evolves. Disulfide bond
formation is another common post-translational modification, where glycans
are covalently attached to proteins. This helps proteins fold properly and
extends their half-life by protecting against proteases. Cell-free
glycosylation is possible but requires careful control and is not cost-efficient.
Disulfide bonds form at the end of the synthesis process, usually in the
endoplasmic reticulum. However, inexpensive methods can produce
disulfide bonds. Protein expression yields non-modified peptides, which lack
post-translational modifications necessary for therapeutic applications. This
can limit the use of cell-free protein expression for developing therapeutic
proteins and pharmaceuticals. (Zawada et al.2022)

6. Conclusion

The drawback of intact cells for gene expression has led to efforts to develop
alternative strategies. An in vitro approach to subunit assembly using
purified components from E. coli cell-free systems has been achieved. The
creation of a new gene expression technology based on fully functionalized
cells has interdisciplinary ramifications and potential spin-offs in various
industries.

E. coli cell-free systems for protein synthesis have been established and are
the beginning of a major advancement in gene expression technology. The
future relies on developing superior cell-free systems. Progress has been
made in using bacterial cell lysates to express eukaryotic proteins and using
defined components for cell-free translation. The use of the eukaryotic
21
HSP70 protein has also been explored for enhancing the heat stability of E.
coli translation. A eukaryotic system (yem) for cell-free protein synthesis
has been reported.

7. References
1. Garenne, D., Haines, M. C., Romantseva, E. F., Freemont, P.,
Strychalski, E. A., & Noireaux, V. (2021). Cell-free gene expression.
Nature reviews methods primers, 1(1), 49.
2. Silverman, A. D., Karim, A. S., & Jewett, M. C. (2020). Cell-free
gene expression: an expanded repertoire of applications. Nature
Reviews Genetics.
3. Sigalova, O. M., Zhao, B., Viales, R. R., Zaugg, J. B., & Furlong, E.
E. (2021). Quantitative analysis of the functional impact of genetic
variation on transcription factor binding during embryogenesis.
INTEGRATING GENETIC, ENVIRONMENTAL AND
DEVELOPMENTAL VARIATION TO STUDY GENE
REGULATORY MECHANISMS, 83.
4. Zawada, J. F., Burgenson, D., Yin, G., Hallam, T. J., Swartz, J. R., &
Kiss, R. D. (2022). Cell-free technologies for biopharmaceutical
research and production. Current Opinion in Biotechnology, 76,
102719.
5. McNally, J. R., Ames, A. M., Admiraal, S. J., & O’Brien, P. J. (2023).
Human DNA ligases I and III have stand-alone end-joining capability,
but differ in ligation efficiency and specificity. Nucleic Acids
Research, 51(2), 796-805.
6. Sharma, S., Kajjo, S., Harra, Z., Hasaj, B., Delisle, V., Ray, D., ... &
Fabian, M. R. (2023). Uncovering a mammalian neural-specific poly

22
 (A) binding protein with unique properties. Genes & Development,
37(15-16), 760-777.
7. Cui, Y., Chen, X., Wang, Z., & Lu, Y. (2022). Cell-free PURE
system: evolution and achievements. BioDesign Research.
8. Peťková, M., Švubová, R., Kyzek, S., Medvecká, V., Slováková, Ľ.,
Ševčovičová, A., & Gálová, E. (2021). The effects of cold
atmospheric pressure plasma on germination parameters, enzyme
activities and induction of DNA damage in barley. International
Journal of Molecular Sciences, 22(6), 2833.
9. Silverman, A. D. (2021). Design and Optimization of a Field-
Deployable Biosensing Platform for Measuring Water Quality.
10.Qiao, X., van der Zanden, S. Y., Wander, D. P., Borràs, D. M., Song,
J. Y., Li, X., ... & Neefjes, J. (2020). Uncoupling DNA damage from
chromatin damage to detoxify doxorubicin. Proceedings of the
National Academy of Sciences, 117(26), 15182-15192.
11.Liu, W. Q., Wu, C., Jewett, M. C., & Li, J. (2020). Cell‐free protein
synthesis enables one‐pot cascade biotransformation in an aqueous‐
organic biphasic system. Biotechnology and Bioengineering, 117(12),
4001-4008.
12.Chiba, C. H., Knirsch, M. C., Azzoni, A. R., Moreira, A. R., &
Stephano, M. A. (2021). Cell-free protein synthesis: Advances on
production process for biopharmaceuticals and immunobiological
products. Biotechniques, 70(2), 126-133.
13.Lafata, K. J., Corradetti, M. N., Gao, J., Jacobs, C. D., Weng, J.,
Chang, Y., ... & Yin, F. F. (2021). Radiogenomic analysis of locally
advanced lung cancer based on CT imaging and intratreatment
changes in cell-free DNA. Radiology: Imaging Cancer, 3(4), e200157.
23
14.Galiñanes Reyes, S. L. (2021). Cell-free protein systems and in vitro
display methods as compelling tools for high-throughput screening.
15.Abdueva, D., Lai, K., Dilger, K., Cunningham, R., Lam, K., Boquiren,
R., ... & Rava, R. (2024). Extracting Regulatory Active Chromatin
Footprint from Cell-Free DNA.
16.Brookwell, A., Oza, J. P., & Caschera, F. (2021). Biotechnology
applications of cell-free expression systems. Life.
17.Hill, C. H., Pekarek, L., Napthine, S., Kibe, A., Firth, A. E., Graham,
S. C., ... & Brierley, I. (2021). Structural and molecular basis for
Cardiovirus 2A protein as a viral gene expression switch. Nature
communications, 12(1), 7166.
18.Cheng, L., De, C., Li, J., & Pertsinidis, A. (2023). Mechanisms of
transcription control by distal enhancers from high-resolution single-
gene imaging. BioRxiv.
19.Wang, W., Peng, X., Jin, Y., Pan, J. A., & Guo, D. (2022). Reverse
genetics systems for SARS‐CoV‐2. Journal of Medical Virology,
94(7), 3017-3031.
20.Wang, H., Fu, T., Du, Y., Gao, W., Huang, K., Liu, Z., ... & Zitnik,
M. (2023). Scientific discovery in the age of artificial intelligence.
Nature, 620(7972), 47-60.
21.Choi, Y. N., Cho, N., Lee, K., Gwon, D. A., Lee, J. W., & Lee, J.
(2023). Programmable Synthesis of Biobased Materials Using Cell‐
Free Systems. Advanced Materials, 35(4), 2203433.
22.Copeland, C. E., Langlois, A., Kim, J., & Kwon, Y. C. (2021). The
cell-free system: A new apparatus for affordable, sensitive, and
portable healthcare. Biochemical Engineering Journal, 175, 108124.

24
23.Li, B., Yang, Y. T., Capra, J. A., & Gerstein, M. B. (2020). Predicting
changes in protein thermodynamic stability upon point mutation with
deep 3D convolutional neural networks. PLoS computational biology,
16(11), e1008291.
24.Sorokina, I., Mushegian, A. R., & Koonin, E. V. (2022). Is protein
folding a thermodynamically unfavorable, active, energy-dependent
process?. International journal of molecular sciences, 23(1), 521.
25.Smolskaya, S., Logashina, Y. A., & Andreev, Y. A. (2020).
Escherichia coli extract-based cell-free expression system as an
alternative for difficult-to-obtain protein biosynthesis. International
Journal of Molecular Sciences, 21(3), 928.

25