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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.