Download Cell Free Protein Synthesis

Cell Free Protein Synthesis
(CFPS), or
In-Vitro Protein Synthesis
By Puria Rafsanjani Nejad
Intro
– Definition:
production of protein using biological machinery without the use of living cells.
– Not constrained by a cell wall or homeostasis conditions necessary to maintain
cell viability
– Direct access and control of the translation environment
– Advantageous for:
optimization of protein complexes, studying protein synthesis, incorporating
non-natural amino acids, high-throughput screens, and synthetic biology.
How is it achieved?
– Exploiting the cellular protein synthesis machinery to direct protein synthesis
outside intact cells.
– Exogenous messenger RNA (mRNA) or DNA as template
Combining:
– Crude lysate from growing cells
which contains all the necessary enzymes and machinery
– Supply of essential amino acids
– Nucleotides
– Salts, Energy-generating factors.
Cellular Protein Synthesis and
its Machinery
Cell-free protein synthesis
system for producing
proteins or (poly)peptidebased materials
CFPS requires cell extract, an energy
regeneration system, and chemical substrates
and salts (e.g., NTPs, amino acids, salts, and
cofactors). Cell-free transcription and translation
is initiated by adding DNA template (plasmid or
PCR-amplified linear DNA templates) into the
CFPS reaction.
Seok Hoon Hong, ” Non-standard amino acid
incorporation into proteins using Escherichia
coli cell-free protein synthesis”, Front. Chem.,
2014
Sequence Info Source
– coupled System, where DNA is used as template
are generally simpler and more efficient; they also avoid problems of mRNA
degradation and mRNA secondary structure
– or as an uncoupled system, which requires mRNA template produced from
native sources or by in vitro transcription.
Uncoupled systems control the amount of input mRNA and can express proteins
in the absence of DTT.
– Cell-free systems can be used to express either a single gene or a DNA library
Coupled Systems
– DNA template may be in the form of a plasmid or polymerase chain reaction
(PCR) fragment
must contain:
– a promoter (T7, SP6 or T3 are most commonly used)
– a translation initiation signal such as a Shine–Dalgarno (prokaryotic) or Kozak
(eukaryotic) sequence
To increase the expression level”
– a transcription and translation
termination region is also required.
Limiting Factors
– Efficiency of the energy supply
– Accumulation of inhibitors
Methods to overcome these limitations:
concentrated batch systems, continuous flow and continuous exchange methods,
the bilayer diffusion system, and systems employing hollow fiber membranes.
– Molecular sizes up to 400 kilo Daltons
Some Proteins produced by CFPS
– various enzymes, growth factors, membrane proteins, protein complexes and
viral capsids
PURE system has been successfully used to express:
– Dihydrofolate reductase, λ-lysozyme, green fluorescent protein (GFP),
glutathione S-transferase and the T7 gene 10 product
– Some integral membrane proteins
Advantages of cell-free protein
synthesis
– Can produce proteins directly from a PCR fragment or an mRNA template without
the need for E. coli cloning, allowing it to be easily adapted for high throughput
protein synthesis
– Can simultaneously express multiple templates, permitting the production of a
protein population in a single reaction
– Often generate soluble and functional proteins
– Is more adjustable and controllable,
– Allows the efficient incorporation of non-natural or chemically-modified amino
acids into the expressed protein at desired positions during translation, thereby
generating novel molecules for proteomic applications.
– Finally, cell-free systems can produce proteins that are not physiologically tolerated
by the living cell— eg toxic, proteolytically sensitive or unstable proteins.
Properties of cell-free
synthesized proteins
– Protein folding:
Nascent proteins have been demonstrated to fold co-translationally on
ribosomes in cell-free systems in a manner similar to protein folding in vivo, ie a
growing peptide starts to fold as it emerges from the large ribosomal subunit or
immediately at the end of translation prior to release from the ribosome
– Molecular chaperones, ribosomes, ribosomal RNA
– Many proteins have been expressed in cell-free systems with correct folding and
processing, yielding active molecules.
– DTT
Folding Proteins That Are Not
Folded Correctly In Vivo
– Many proteins that fail to be functionally produced by cell-based methods can
be actively expressed in a cell-free system with defined conditions, indicating
that cell-free expression may be a more suitable system for producing folded
proteins !!!
In Vitro Expression Cloning
(IVEC)
– Cell-free systems have led to the establishment of a routine functional
screening method known as in vitro expression cloning (IVEC).
– a large complementary DNA (cDNA) library is broken down into pools of 50–100
clones of plasmid templates. The pools are expressed in a coupled cell free
system and screened for function
– IVEC has been used successfully to clone and identify enzymes, protein
substrates, phospholipid-binding proteins and a sister chromatid separation
inhibitor.
Protein-Producing Nanoparticles
– What if
the machinery required to achieve transcription and
translation is we encapsulated inside of artificial
membranes?
– cDNA was used to eliminate complexities associated with mRNA splicing.
– To form vesicles, a phospholipid that selfassembles into soft lipid bilayers at
physiological temperature was used (1,2-dimyristoyl-sn-glycero-3phosphocholine(DMPC); a phosphatidylcholine with two 14-carbon tails).
Formation of Particles
– Microscale vesicles were formed spontaneously after mixing the extract and
DNA with DMPC.
– The particles were collected by centrifugation, and non-entrapped extract, DNA,
and protein were removed by repeated washing.
Proteins Produced
– The lipid vesicles were capable of producing GFP,
– To test if the particles were capable of producing proteins that are
enzymatically active, a template encoding Renilla luciferase was incorporated
into the particles
– When luciferin was added to the lysate, the solution emitted robust
luminescence, indicating that the luciferase produced in the particles was
enzymatically active.
How Small Can These NPs be?
– Particles with average diameters of ∼400, 250, and 170 nm were all capable of
producing functional luciferase;
– however, 100 nm particles were not. CryoTEM images suggest that the 170 nm
extruded nanoparticles display an elliptical morphology with the ribosomes and
code inside.
– Testing the internal content ofthe 100 nm particles showed they lack DNA
Which size is more efficient?
– Interestingly, when comparing the total amount of protein produced,
dispersions with smaller particles (170 or 250 nm) produced more protein than
dispersions with larger (400 nm)
– the exact reason for this phenomenon is unclear but it seems:
possible that the close proximity of the “reacting” components To the
machinery plays a role in more efficient use of resources during these processes
Triggering
– To control the production of proteins temporally, a photolabile protecting group
was conjugated to the DNA.
– Luciferaseencoding DNA was caged with
1-(4,5-dimethoxy-2-nitrophenyl)
diazoethane (DMNPE) to block
transcription.
– UV irradiation at 365 nm uncaged the
DNA, and luciferase was produced.
Radiation timing
In vivo
benefits
– Such an approach, in which autonomous nanoscale production units are
located in the body and can be remotely activated to synthesize a potent
compound from inert precursors, may find utility in the localized delivery of
Therapeutics
– To date, this objective has been met for therapeutic applications only with live
bacteria that were predesigned to produce proteins in disease sites.
– Unlike bacterial systems, artificial systems are modular, and their
physical/chemical properties can be modified.